WO2022191130A1 - Method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent - Google Patents

Method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent Download PDF

Info

Publication number
WO2022191130A1
WO2022191130A1 PCT/JP2022/009718 JP2022009718W WO2022191130A1 WO 2022191130 A1 WO2022191130 A1 WO 2022191130A1 JP 2022009718 W JP2022009718 W JP 2022009718W WO 2022191130 A1 WO2022191130 A1 WO 2022191130A1
Authority
WO
WIPO (PCT)
Prior art keywords
cardiomyocytes
cardiomyocyte
inhibitors
cells
inhibitor
Prior art date
Application number
PCT/JP2022/009718
Other languages
French (fr)
Japanese (ja)
Inventor
潤 山下
美樹 吉岡
Original Assignee
国立大学法人京都大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人京都大学 filed Critical 国立大学法人京都大学
Priority to JP2023505544A priority Critical patent/JPWO2022191130A1/ja
Publication of WO2022191130A1 publication Critical patent/WO2022191130A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for proliferating and producing cardiomyocytes and a cardiomyocyte proliferating agent used therein.
  • Non-Patent Document 1 by the present inventors has already identified and reported a compound cocktail that induces proliferation of mouse ES cells and human iPS cell-derived cardiomyocytes.
  • Non-Patent Document 1 the present inventors have found a technique for inducing the proliferation of mouse ES cell- and human iPS cell-derived cardiomyocytes.
  • mouse and human myocardial cells differ in their proliferation-arrested state, suggesting that there is a mechanism specific to human cells. Therefore, further elucidation of the human cell-specific mechanism and discovery of the method and necessary factors for proliferation of human cardiomyocytes are expected to greatly contribute to cardiac regenerative medicine.
  • the present invention has been made in view of the above circumstances, and provides a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a method for producing cardiomyocytes, which enables induction of proliferation of human cardiomyocytes with a drug and contributes to cardiac regenerative medicine.
  • An object of the present invention is to provide a cardiomyocyte proliferation agent.
  • Embodiments of the invention have the following aspects.
  • proliferation of cardiomyocytes comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors; Method.
  • the above method for proliferation of cardiomyocytes wherein the compound is an ALK inhibitor.
  • the ALK inhibitor is a TGF ⁇ RI (ALK5) inhibitor.
  • ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • cardiomyocyte production comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Method.
  • ALK inhibitors cytochrome P450 omega-hydroxylase inhibitors
  • fatty acid synthesis inhibitors fatty acid synthesis inhibitors.
  • ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • the method for producing cardiomyocytes, wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes.
  • the above production method wherein the pluripotent stem cells are derived from humans.
  • a cardiomyocyte proliferation agent containing, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  • the cardiomyocyte proliferation agent, wherein the compound is an ALK inhibitor.
  • the cardiomyocyte proliferation agent wherein the ALK inhibitor is a TGF ⁇ RI (ALK5) inhibitor.
  • the cardiomyocyte proliferation agent wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • the cardiomyocyte proliferation agent wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes.
  • the cardiomyocyte proliferation agent, wherein the pluripotent stem cells are human-derived.
  • a method for proliferating cardiomyocytes a method for producing cardiomyocytes, and a cardiomyocyte-proliferating agent that can induce the proliferation of human cardiomyocytes and contribute to cardiac regenerative medicine can be obtained.
  • FIG. 3 is a schematic diagram showing an operation for inducing differentiation of cardiomyocytes in this example.
  • FIG. 2 is a graph showing cTnT(+)% of cardiomyocytes and a schematic diagram of a cardiomyocyte culture plate in this example.
  • Fig. 2 is a schematic diagram showing the operation of proliferation evaluation for selection of a positive control in this example.
  • FIG. 4 is a photograph showing cell staining of each positive control compound in this example.
  • FIG. 4 is a graph showing the amount of S-phase cells when each positive control compound of this example was added.
  • FIG. 10 is a graph showing the amount of M-phase cells when each positive control compound of this example was added.
  • FIG. 4 is a schematic diagram showing the operation of cardiomyocyte proliferation in this example.
  • FIG. 3 is a photographic diagram showing immunostaining of cardiomyocyte proliferation in this example.
  • FIG. 4 is a graph showing cell number evaluation of cardiomyocyte proliferation in this example.
  • FIG. 4 is a graph showing the EdU+/cTnT+ nuclei counted for the compounds picked up in the 1st screening of this example.
  • FIG. 10 is a graph showing the number of EdU+/cTnT+ nuclei counted for another compound picked up in the 1st screening of this example.
  • FIG. 10 is a graph showing counts of EdU+/cTnT+ nuclei for yet another compound picked up in the 1st screening of this example.
  • FIG. 3 is a photographic diagram showing a part of immunostaining that was the basis for counting in this example.
  • FIG. 2 is a photographic diagram showing a bright field after culturing for 1 day and 1+5 days in the cell number evaluation test of this example.
  • FIG. 4 is a graph showing evaluation of total cell count in tests using various ALK5 inhibitors of this example.
  • FIG. 4 is a graph showing evaluation of cTnT(+)% in tests using various ALK5 inhibitors of this example.
  • FIG. 4 is a graph showing evaluation of cTnT(+) cell numbers in tests using various ALK5 inhibitors of this example.
  • FIG. 2 is a schematic diagram showing one mode of operation for inducing differentiation of cardiomyocytes.
  • cardiomyocyte proliferation method the cardiomyocyte production method, and the cardiomyocyte proliferation agent according to the present invention will be described with reference to embodiments.
  • the present invention is not limited to the following embodiments.
  • cardiomyocytes are cultured in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Including process.
  • ALK inhibitor broadly refers to a component that inhibits the function of ALK (activin receptor-like kinase).
  • ALK inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds.
  • the ALK inhibitor is preferably a TGF ⁇ RI (TGF- ⁇ receptor type-1) (ALK5) inhibitor.
  • the ALK5 inhibitor is preferably one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • a cytochrome P450 omega-hydroxylase inhibitor broadly refers to a component that inhibits the function of cytochrome P450 omega-hydroxylase.
  • Cytochrome P450 omega-hydroxylase inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds.
  • a preferred cytochrome P450 omega-hydroxylase inhibitor is 12(S)-hydroxy-16-heptadecinoic acid.
  • Fatty acid synthesis inhibitors broadly refer to ingredients that inhibit fatty acid synthesis.
  • Fatty acid synthesis inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds.
  • the fatty acid synthesis inhibitor is C75 or cerulenin.
  • ALK inhibitors cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  • concentration of the medium is 0.5 to 40 ⁇ M, preferably 2 to 10 ⁇ M.
  • cardiomyocytes broadly refer to myocardial cells, specifically cells having self-pulsating properties. Cardiomyocytes also include myocardial progenitor cells. Cardiomyocytes also broadly include cells that have the potential to give rise to cardiomyocytes and vascular smooth muscle that form beating muscle and electrically conductive tissue. Cardiomyocytes and cardiomyocyte progenitor cells may be mixed with each other or may be individual. Cardiomyocytes and myocardial progenitor cells broadly include cells positive for the myocardial marker cardiac troponin (cTNT) or ⁇ -myosin heavy chain ( ⁇ MHC). Cardiomyocytes also include cell populations that contain a high percentage of cardiomyocytes relative to other cell types. A cell population containing 50% or more cardiomyocytes is preferred, preferably 60% or 70% or more, more preferably 80% or more, and particularly preferably 90% or more.
  • cTNT myocardial marker cardiac troponin
  • ⁇ MHC ⁇ -myosin heavy chain
  • the cardiomyocytes are preferably cardiomyocytes derived from pluripotent stem cells.
  • ES cells, ntES cells, GS cells, EG cells, Muse cells, iPS cells, and the like are known as pluripotent stem cells.
  • iPS cells are preferably used as the pluripotent stem cells of the present embodiment.
  • iPS cells are pluripotent stem cells derived from somatic cells and have pluripotency and self-renewal potential.
  • Pluripotency means the ability to differentiate into all three germ layer lineages.
  • the self-renewal ability means the ability to proliferate while maintaining an undifferentiated state.
  • the pluripotent stem cells are more preferably human-derived pluripotent stem cells.
  • human-derived iPS cells Human iPS cells, HiPS cells, human iPS cells
  • a method for obtaining iPS cells can be performed by a conventionally known method.
  • K. Takahashi and S.; Yamanaka (2006) Cell, 126:663-676, or the method described in WO2007/069666 or the like can be used.
  • it can be produced by introducing a specific reprogramming factor into a somatic cell in the form of DNA or protein.
  • Reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERAs, ECAT15-2, Tcl1, Genes such as beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, or proteins encoding them can be used singly or in combination.
  • the reprogramming factor is DNA, it can be introduced into cells using vectors such as viruses, plasmids or artificial chromosomes, lipofection, liposomes, or microinjection.
  • the reprogramming factor is a protein, it can be introduced into cells by techniques such as lipofection, fusion with a cell membrane permeable peptide, or microinjection.
  • a method for obtaining pluripotent stem cell-derived cardiomyocytes that is, induction of differentiation of cardiomyocytes from pluripotent stem cells can be performed by a known method. See, for example, Toyama, S.; , et al. (2013). Cell Stem Cell 12(1):127-137, Laflamme et al. , Nat Biotech. 2007 Sep;25(9):1015-24, Uosaki et al. , Plos ONE. 2011; 6(8): e23657 can be used.
  • a ready-made myocardial induction medium, an existing kit containing the medium, or the like can also be used.
  • various myocardial differentiation-inducing factors can be used to induce the differentiation of pluripotent stem cells into cardiomyocytes.
  • Cardiac differentiation inducers include mesoderm-inducing factors (activin A, BMP4, bFGF, VEGF, or SCF, etc.), cardiac determinants (VEGF, or DKK1, etc.), Wnt signal inhibitors (XAV939, IWR-1, IWP- 2, or IWP-4, etc.), BMP signal inhibitors (NOGGIN, etc.), TGF ⁇ /activin/NODAL signal inhibitors (SB431542, etc.), retinoic acid signal inhibitors, or cardiac differentiation factors (e.g., VEGF, bFGF, or DKK, etc.). These myocardial differentiation-inducing factors can be used singly or in combination.
  • pluripotent stem cells are cultured to induce differentiation into mesoderm.
  • Differentiation into mesoderm can be induced by culturing for 1 to 7 days in the presence of the mesoderm-inducing factor.
  • culture is performed for 1 to 4 days in the presence of the cardiomyocyte differentiation-inducing factor.
  • culture is preferably performed for 2 to 3 days in the presence of cardiomyocyte differentiation-inducing factors.
  • Dkk-1 which is an endogenous Wnt inhibitor
  • XAV939 and IWP-4 which are Wnt signal inhibitors
  • myocardial differentiation-inducing factors may be used in combination, for example, XAV939 and IWP-4 may be used in combination.
  • culture is performed in the presence of matrigel for 1 to 2 days as a pretreatment before inducing differentiation into mesoderm, and culture is further performed in the presence of bFGF when inducing differentiation into mesoderm. is also preferred. By this operation, cardiomyocyte differentiation is induced with higher efficiency, and pulsating cells appear from around 4 to 10 days after the initiation of culture for inducing differentiation into mesoderm.
  • the cardiomyocyte proliferation method of the present embodiment includes the step of culturing the cardiomyocytes in the presence of any one or more of the compounds.
  • the medium may contain other components in addition to the above components.
  • Cultivation for proliferation of cardiomyocytes may be carried out by separating the cardiomyocytes obtained by the aforementioned induction of differentiation, transplanting them to a plate, and culturing them. Alternatively, culture for proliferation may be performed again on the plate that has been cultured for differentiation induction.
  • the cardiomyocytes obtained by the induction of differentiation are preferably seeded again at a low density and cultured to proliferate the cardiomyocytes.
  • the seeding density can be appropriately selected from approximately 0.1 to 5 ⁇ 10 4 cells/cm 2 . Preferably, it is 0.5 to 2.0 ⁇ 10 4 cells/cm 2 .
  • Culture for proliferation of cardiomyocytes can be performed by conventionally known means and conditions.
  • the culture temperature is 30 to 40° C., preferably about 37° C.
  • the CO 2 concentration is about 2% to 5%
  • the O 2 concentration is so-called hypoxic conditions, eg about 5% to 20%.
  • the medium used for culturing cardiomyocytes is not particularly limited, and can be appropriately selected according to the purpose.
  • a basal medium can be used.
  • basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, and RPMI 1640 medium. , Fischer's medium, Neurobasal Medium (Life Technologies), StemPro34 (Invitrogen), and a mixed medium containing these may be used.
  • the basal medium can be serum-containing or serum-free. When containing serum, a basal medium containing 5% to 20% by weight of serum can be used.
  • the medium may contain other ingredients.
  • Other ingredients include nutrients and other additives.
  • Nutrients include, for example, serum replacement.
  • Serum substitutes include albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace amounts It may contain one or more elements such as 2-mercaptoethanol or 1-thiolglycerol.
  • Other ingredients include lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, or inorganic salts. It may contain one or more kinds.
  • RPMI medium is used as a medium and cultured in the presence of insulin as an antibiotic.
  • the cardiomyocytes obtained by the cardiomyocyte proliferation method of the present embodiment can be used for treatment of human heart disease (cardiac disease).
  • the heart disease broadly includes diseases in the heart or diseases requiring treatment of the heart.
  • the obtained cardiomyocytes may be administered to a patient, or the obtained cardiomyocytes may be used as a therapeutic agent for heart disease.
  • a method for administering the myocardial cells a means of suspending the cells in a liquid and administering them to the myocardium, or attaching the cells via a sheet, bandage, or the like can be used.
  • the heart disease includes heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic phase hypertrophic cardiomyopathy, dilated cardiomyopathy, etc., or defects due to disorders. It is not limited as long as it is a disease affecting the heart.
  • cardiomyocytes are cultured in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Including process.
  • the compound and the step of culturing cardiomyocytes can be selected from those described in the cardiomyocyte proliferation method. That is, the method for producing cardiomyocytes of the present embodiment preferably includes the steps in the method for growing cardiomyocytes. In other words, by adding the steps in the method for growing cardiomyocytes to cultured cardiomyocytes, the cardiomyocytes proliferate, and the cardiomyocytes for the proliferation can be produced.
  • the cardiomyocyte proliferation agent of this embodiment contains, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  • the compound can be selected from those described in the cardiomyocyte proliferation method.
  • the cardiomyocyte-proliferating agent of the present embodiment may be used in the above-described cardiomyocyte-proliferating method and cardiomyocyte-producing method. That is, it may be added to proliferate cultured cardiomyocytes.
  • the cardiomyocyte-proliferating agent of the present embodiment is used in a cardiomyocyte-proliferating method and a cardiomyocyte-producing method
  • the cardiomyocyte-proliferating agent of the present embodiment is added to the culture system of cardiomyocytes in culture. can be used.
  • the cardiomyocyte proliferation agent may be administered to the patient.
  • the cardiomyocytes are preferably those in the patient's body.
  • Cardiac diseases or conditions requiring treatment of the heart include the examples described in the cardiomyocyte proliferation method.
  • the cardiomyocyte proliferation agent may contain other ingredients as appropriate.
  • it may contain ingredients conventionally known as those used in the treatment of diseases of the heart and other circulatory systems.
  • Cardiomyocyte proliferation agents including ALK inhibitors, may also be used for anti-cancer therapeutic effects.
  • an ALK inhibitor In the cardiomyocyte proliferation method, cardiomyocyte production method, and cardiomyocyte proliferation agent of the present embodiment, an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, or a fatty acid synthesis inhibitor is used. These compounds have the effect of inducing proliferation of human cardiomyocytes.
  • the present inventors conducted compound screening using human iPS cell-derived cardiomyocytes in order to discover compounds that can induce proliferation of human cardiomyocytes.
  • compound library screening using human myocardial cells the above compound group having myocardial proliferation activity was found.
  • the same compound was able to induce mitosis twice or more even in human cardiomyocytes cultured for a long period of about 3 months.
  • some cardiomyocytes do not show reactivity to the same compound, and we are currently clarifying the difference between the two by comprehensive gene expression analysis.
  • the same compound did not exhibit a significant myocardial proliferative effect on mouse ES cell-derived cardiomyocytes, suggesting the existence of a human cardiomyocyte-specific growth control mechanism.
  • ALK inhibitors increased the number of human cardiomyocytes by about 1.5-2 fold. Three divisions have been confirmed in cardiomyocytes that respond well to ALK inhibitors. That is, it corresponds to proliferation to eight times the number of cells before division. The effects of ALK inhibitors are human cell-specific and weak in mouse myocardial cells. There has been no report on cardiomyocyte proliferation control by ALK inhibitors. In addition, among ALK inhibitors, there are compounds that have already been tested as anticancer agents, and their use as pharmaceuticals is possible.
  • the method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the cardiomyocyte-proliferating agent of the present embodiment elucidate the molecular mechanism of myocardial proliferation and responsiveness to proliferation stimuli, and enable human cardiomyocytes to proliferate. It is expected to contribute to the development of regenerative medicine.
  • the present inventors added matrigel on day-1 and bFGF on day 1-5 to the two-dimensional cardiomyocyte differentiation induction method DD-protocol (Laflamme et al, Nat Biotech, 2007, etc. described above), A system for more efficient myocardial differentiation (Uosaki et al, Plos ONE, 2011, etc. mentioned above) has been established (Fig. 19). In this method, Dkk-1 was added in the presence of RPMI medium+B27 on days 5-7 to differentiate into cardiomyocytes.
  • This differentiation system can be further manipulated to purify only cells expressing a certain marker ( ⁇ -type platelet-derived growth factor receptor; PDGFR ⁇ ) (Fukushima et al, PLoS One. 2020 Nov 2; 15 ( 11): e0241287.), this purification procedure was carried out in the examples below.
  • a certain marker ⁇ -type platelet-derived growth factor receptor; PDGFR ⁇
  • Fig. 1 shows an outline of the operation for inducing the differentiation of cardiomyocytes used in this example in chronological order.
  • Human iPS cells (201B6) provided by Professor Shinya Yamanaka of Kyoto University were used as iPS cells.
  • bFGF addition amount: 4 ng/mL, Final Conc.
  • matrigel addition amount: 1/1 of the medium was added from day -1 to day 0. 60 was further added.
  • FIG. 2 shows the cardiac troponin T expression rate (cTnT(+)%) of cells obtained by this system. It was shown that this differentiation system yields cardiomyocytes with a cTnT(+)% of over 97% (graph on the left side of FIG. 2). The cardiomyocytes were plated again at a lower density and used for subsequent analysis (schematic diagram on the right side of FIG. 2).
  • FIG. 3 shows a schematic diagram of proliferation evaluation (from Patent Document 1) for selection of a positive control.
  • a positive control for proliferation evaluation a compound found by the present inventors in a mouse cardiomyocyte proliferation screening system was set as a reference.
  • CHIR means CHIR99021.
  • FIG. 4 shows cell staining of DMSO (control), BIO, SU1498, and KN93-administered cultures.
  • BIO, SU1498, and KN93 cardiomyocytes (cTnT) dark in the photograph were stained with EdU and DAPI, which were bright in the photograph.
  • FIG. 5 shows the cell amount in S phase (EdU-positive CM), and
  • FIG. 6 shows the cell amount in M phase (pH 3, ie, phospho-histone positive).
  • BIO, SU1498 and KN93 were found to have myocardial proliferation ability.
  • BIO and CHIR99021 which are GSK3 ⁇ inhibitors
  • SB203580 which is a p38MAPK inhibitor (p38i)
  • SU1498 which is an ERK activator
  • FIG. 7 shows the flow of proliferation of cardiomyocytes.
  • Cardiomyocytes (d19) induced from human iPS cells were seeded under low-density conditions (1.33 ⁇ 10 4 cells/cm 2 ). After culturing in the presence of serum for 24 hours, adhesion of cardiomyocytes to the plate was confirmed, and each compound of the positive control was added at a desired concentration. After culturing for 2 days, 20 ⁇ M of EdU was added. After culturing for another day, the cells were fixed and immunostained with cardiac troponin T (cTnT), which is a cardiomyocyte marker.
  • cTnT cardiac troponin T
  • FIG. EdU and DAPI light color in the photograph
  • cTnT dark color in the photograph
  • DAPI signals arrows, respectively
  • Addition of the positive control showed a more than 1.5-fold increase in the number of EdU+/cTnT+ cell nuclei.
  • Bio-active lipid screening library I & II were analyzed.
  • Bio-active lipid screening library I used more than 785 compounds to be screened stored in 96-well matrix tubes in 1.0 mM DMSO.
  • Bio-active lipid screening library II used 96-well matrix tubes containing >185 compounds to be screened in 0.1 mM DMSO.
  • Cell seeding conditions ⁇ Myocardial muscle induced to differentiate from hiPS cells was used. ⁇ Cell seeding density 1.33 ⁇ 10 4 cells/cm 2 - Reagent was added on the day after cell seeding (20 hrs later). Evaluation conditions: ⁇ cTnT(+)/EdU(+) nuclear numbers -Fixed and stained 3 days (72 hrs) after the addition of the compound. -EdU uptake was evaluated at 24 hrs.
  • Compound conditions ⁇ Cayman Bioactive lipid I & II screening library plate 1-11; 10 ⁇ M, 2 ⁇ M (stock; 1.0 mM) plate 12-14; 1 ⁇ M, 0.2 ⁇ M (stock; 0.1 mM)
  • FIG. 13 shows typical immunostained photographs from which the EdU+/cTnT+ cell nuclei were counted above. From the photograph, addition of each compound (C75, LY364947, 12(S)-hydroxy-16-Hepatadecynoic acid, Cerulenin, SB431542) tends to increase the EdU+/cTnT+ nuclear numbers (bright signal) compared to the negative control. was shown to be in
  • EdU+/cTnT+ nuclear numbers were used as evaluation indices.
  • EdU+/cTnT+ indicates cardiomyocytes undergoing DNA synthesis during the period of EdU addition (24 hrs).
  • human cardiomyocytes are mostly mononuclear cells, but there are also binuclear cells that are temporarily dividing and have binuclear cells. Therefore, an increase in EdU+/cTnT+nuclear numbers does not necessarily correspond to an increase in the number of myocardial cells. Therefore, we set up conditions for a system that evaluates the number of myocardial cells.
  • Cardiomyocytes induced to differentiate from hiPS were seeded at a low density (0.93 ⁇ 10 4 cells/cm 2 ) in the same manner as during screening, and after confirming cell adhesion, each of the above compounds was added. Cardiomyocyte numbers were counted after 1 day and 1+5 days. In addition, cTNT FACS analysis was performed.
  • Fig. 15 shows bright field photographs after 1 day and 1+5 days of culture.
  • the negative control in the figure had a cTnT(+) value of 93.8%, and the positive control had a cTnT(+) value of 97.5%. It can be seen that cardiomyocytes tend to increase with the addition of the positive control.
  • ALK5 inhibitors were added to analyze whether the cardiomyocyte number increased.
  • Both LY364947 and SB431542 are ALK5 inhibitors and, as mentioned above, increase the number of myocardial cells. This increase in cardiomyocyte number may be due to inhibition of ALK5 signaling. Therefore, analysis was performed using other ALK5 inhibitors, A83-01 and RepSox.
  • A83-01 is a potent inhibitor of ALK5 and is also known to inhibit ALK4,7.
  • RepSox (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) (denoted as “TGFbR KI” in FIGS. 16-18) ALK5 selective inhibitor.
  • the method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the agent for proliferating cardiomyocytes of the present invention enable the induction of human cardiomyocyte proliferation with drugs, and can contribute to cardiac regenerative medicine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

Provided are a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a cardiomyocyte proliferating agent capable of contributing to cardiac regenerative medicine, that make it possible to induce proliferation of human cardiomyocytes by a drug. The method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent, including a step for culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega hydroxylase inhibitors, and fatty acid synthesis inhibitors

Description

心筋細胞の増殖方法、心筋細胞の製造方法および心筋細胞増殖剤Method for Proliferating Cardiomyocytes, Method for Producing Cardiomyocytes, and Agent for Proliferating Cardiomyocytes
　本発明は、心筋細胞を増殖、製造する方法およびそれに用いる心筋細胞増殖剤に関する。
　本願は、２０２１年３月８日に米国に仮出願された米国特許出願番号６３／１５７，８３４号に基づき優先権を主張し、その内容をここに援用する。 TECHNICAL FIELD The present invention relates to a method for proliferating and producing cardiomyocytes and a cardiomyocyte proliferating agent used therein.
This application claims priority to U.S. Patent Application Serial No. 63/157,834 filed March 8, 2021 in the U.S., the contents of which are hereby incorporated by reference.
　現在、日本では心筋梗塞の症例が年間１０万例弱認められる。心臓の治療において、ヒトの心臓組織が傷害後も修復可能となれば、心筋梗塞による死を激減させうる可能性がある。しかしながら、ヒトの心臓は原則として再生しない臓器とされている。例えば、ゼブラフィッシュや生後１週間のマウスの心筋は、増殖能を保持しており、心臓損傷後も再生が可能であるが、ヒト心筋細胞は増殖能が低く、ターンオーバーは年に数％程度であり、心臓再生能もほぼないと考えられている。 Currently, there are nearly 100,000 cases of myocardial infarction in Japan each year. In cardiac therapy, the ability of human heart tissue to repair after injury could dramatically reduce mortality from myocardial infarction. However, in principle, the human heart is considered an organ that does not regenerate. For example, the myocardium of zebrafish and 1-week-old mice retains proliferative ability and can regenerate after heart injury, but human cardiomyocytes have low proliferative ability and turnover is only a few percent per year. It is thought that heart regenerative capacity is almost non-existent.
　心臓の再生能力の有無は、心筋細胞の増殖能の有無によって決定されているのではないかとの説に則って、心筋細胞を増殖可能とすることにより、心臓の再生が可能になるのでは、と期待され、種々の研究がなされている。しかし、これまでのところ十分に有効と考えられる方法は確立されていない。特に多くの報告はマウス心筋細胞や心臓に関して示されているものであり、増殖停止機構が異なる可能性があるヒト細胞での知見は乏しい。 Based on the theory that the ability of the heart to regenerate is determined by the ability of cardiomyocytes to proliferate, it may be possible to regenerate the heart by enabling the proliferation of cardiomyocytes. This is expected, and various studies are being conducted. However, no method has been established so far that is considered to be sufficiently effective. In particular, many reports have been shown on mouse myocardial cells and hearts, and there is little knowledge on human cells, which may have different growth arrest mechanisms.
　本発明者らは、マウスおよびヒト多能性幹細胞から高効率で心筋細胞を誘導することに成功しており、これら心筋細胞の増殖を誘導する化合物の探索・同定を以前から進めてきた。
　本発明者らによる非特許文献１では、マウスＥＳ細胞およびヒトｉＰＳ細胞由来心筋細胞の増殖を誘導する化合物カクテルをすでに同定し報告している。 The present inventors have succeeded in inducing cardiomyocytes from mouse and human pluripotent stem cells with high efficiency, and have been searching for and identifying compounds that induce proliferation of these cardiomyocytes.
Non-Patent Document 1 by the present inventors has already identified and reported a compound cocktail that induces proliferation of mouse ES cells and human iPS cell-derived cardiomyocytes.
Ｕｏｓａｋｉ　ｅｔ　ａｌ．，Ｃｉｒｃ　Ｃａｒｄｉｏｖａｓｃ　Ｇｅｎｅｔ．，２０１３；６：６２４－６３３．Uosaki et al. , Circ Cardiovasc Genet. , 2013; 6:624-633.
　上述のように、本発明者らは非特許文献１で、マウスＥＳ細胞およびヒトｉＰＳ細胞由来心筋細胞の増殖を誘導する技術を見出している。しかしながら、マウスとヒト心筋細胞では増殖が停止している状況が異なり、ヒト細胞に特異的なメカニズムがあると考えられる。よって、さらにヒト細胞特異的機構の解明を行い、ヒトの心筋細胞の増殖の方法および必要因子を見出すことで、心臓再生医療に大きく寄与できることが期待される。 As described above, in Non-Patent Document 1, the present inventors have found a technique for inducing the proliferation of mouse ES cell- and human iPS cell-derived cardiomyocytes. However, mouse and human myocardial cells differ in their proliferation-arrested state, suggesting that there is a mechanism specific to human cells. Therefore, further elucidation of the human cell-specific mechanism and discovery of the method and necessary factors for proliferation of human cardiomyocytes are expected to greatly contribute to cardiac regenerative medicine.
　本発明は、上記事情に鑑みてなされたものであって、薬剤によりヒト心筋細胞の増殖の誘導を可能とし、心臓再生医療に寄与することのできる心筋細胞の増殖方法、心筋細胞の製造方法および心筋細胞増殖剤を提供することを目的とする。 The present invention has been made in view of the above circumstances, and provides a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a method for producing cardiomyocytes, which enables induction of proliferation of human cardiomyocytes with a drug and contributes to cardiac regenerative medicine. An object of the present invention is to provide a cardiomyocyte proliferation agent.
　本発明の実施態様は、以下の側面を有する。
［１］　ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物の存在下で、心筋細胞を培養する工程を含む、心筋細胞の増殖方法。
［２］　前記化合物がＡＬＫ阻害剤である、前記の心筋細胞の増殖方法。
［３］　前記ＡＬＫ阻害剤がＴＧＦβＲＩ（ＡＬＫ５）阻害剤である、前記の心筋細胞の増殖方法。
［４］　前記ＡＬＫ５阻害剤が、ＬＹ３６４９４７、ＳＢ４３１５４２、Ａ８３－０１、およびＲｅｐＳｏｘからなる群より選択される１以上の化合物である、前記の心筋細胞の増殖方法。
［５］　前記心筋細胞が多能性幹細胞由来の心筋細胞である、前記の心筋細胞の増殖方法。
［６］　前記多能性幹細胞がヒト由来である、前記の心筋細胞の増殖方法。
［７］　ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物の存在下で、心筋細胞を培養する工程を含む、心筋細胞の製造方法。
［８］　前記化合物がＡＬＫ阻害剤である、前記の心筋細胞の製造方法。
［９］　前記ＡＬＫ阻害剤がＴＧＦβＲＩ（ＡＬＫ５）阻害剤である、前記の心筋細胞の製造方法。
［１０］　前記ＡＬＫ５阻害剤が、ＬＹ３６４９４７、ＳＢ４３１５４２、Ａ８３－０１、およびＲｅｐＳｏｘからなる群より選択される１以上の化合物である、前記の心筋細胞の製造方法。
［１１］　前記心筋細胞が多能性幹細胞由来の心筋細胞である、前記の心筋細胞の製造方法。
［１２］　前記多能性幹細胞がヒト由来である、前記の製造方法。
［１３］　ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物を有効成分とする、心筋細胞増殖剤。
［１４］　前記化合物がＡＬＫ阻害剤である、前記の心筋細胞増殖剤。
［１５］　前記ＡＬＫ阻害剤がＴＧＦβＲＩ（ＡＬＫ５）阻害剤である、前記の心筋細胞増殖剤。
［１６］　前記ＡＬＫ５阻害剤が、ＬＹ３６４９４７、ＳＢ４３１５４２、Ａ８３－０１、およびＲｅｐＳｏｘからなる群より選択される１以上の化合物である、前記の心筋細胞増殖剤。
［１７］　前記心筋細胞が多能性幹細胞由来の心筋細胞である、前記の心筋細胞増殖剤。
［１８］　前記多能性幹細胞がヒト由来である、前記の心筋細胞増殖剤。 Embodiments of the invention have the following aspects.
[1] proliferation of cardiomyocytes, comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors; Method.
[2] The above method for proliferation of cardiomyocytes, wherein the compound is an ALK inhibitor.
[3] The above method for growing cardiomyocytes, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.
[4] The above method for proliferation of cardiomyocytes, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
[5] The method for growing cardiomyocytes, wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes.
[6] The method for growing cardiomyocytes, wherein the pluripotent stem cells are human-derived.
[7] Cardiomyocyte production, comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Method.
[8] The method for producing cardiomyocytes, wherein the compound is an ALK inhibitor.
[9] The method for producing cardiomyocytes, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.
[10] The method for producing cardiomyocytes, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
[11] The method for producing cardiomyocytes, wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes.
[12] The above production method, wherein the pluripotent stem cells are derived from humans.
[13] A cardiomyocyte proliferation agent containing, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
[14] The cardiomyocyte proliferation agent, wherein the compound is an ALK inhibitor.
[15] The cardiomyocyte proliferation agent, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.
[16] The cardiomyocyte proliferation agent, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
[17] The cardiomyocyte proliferation agent, wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes.
[18] The cardiomyocyte proliferation agent, wherein the pluripotent stem cells are human-derived.
　本発明によれば、薬剤によりヒト心筋細胞の増殖の誘導を可能とし、心臓再生医療に寄与することのできる心筋細胞の増殖方法、心筋細胞の製造方法および心筋細胞増殖剤が得られる。 According to the present invention, a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a cardiomyocyte-proliferating agent that can induce the proliferation of human cardiomyocytes and contribute to cardiac regenerative medicine can be obtained.
本実施例の心筋細胞の分化誘導の操作を示す模式図である。FIG. 3 is a schematic diagram showing an operation for inducing differentiation of cardiomyocytes in this example. 本実施例の心筋細胞のｃＴｎＴ（＋）％を示すグラフ図および心筋細胞培養プレートの模式図である。FIG. 2 is a graph showing cTnT(+)% of cardiomyocytes and a schematic diagram of a cardiomyocyte culture plate in this example. 本実施例のポジティブコントロールの選定のための増殖評価の操作を示す模式図である。Fig. 2 is a schematic diagram showing the operation of proliferation evaluation for selection of a positive control in this example. 本実施例のポジティブコントロール各化合物の細胞染色を示す写真図である。FIG. 4 is a photograph showing cell staining of each positive control compound in this example. 本実施例のポジティブコントロール各化合物を添加した際のＳ期の細胞量を示すグラフ図である。FIG. 4 is a graph showing the amount of S-phase cells when each positive control compound of this example was added. 本実施例のポジティブコントロール各化合物を添加した際のＭ期の細胞量を示すグラフ図である。FIG. 10 is a graph showing the amount of M-phase cells when each positive control compound of this example was added. 本実施例の心筋細胞の増殖の操作を示す模式図である。FIG. 4 is a schematic diagram showing the operation of cardiomyocyte proliferation in this example. 本実施例の心筋細胞の増殖の免疫染色を示す写真図である。FIG. 3 is a photographic diagram showing immunostaining of cardiomyocyte proliferation in this example. 本実施例の心筋細胞の増殖の細胞数評価を示すグラフ図である。FIG. 4 is a graph showing cell number evaluation of cardiomyocyte proliferation in this example. 本実施例の１ｓｔ　ｓｃｒｅｅｎｉｎｇでピックアップした化合物に対するＥｄＵ＋／ｃＴｎＴ＋核数をカウントしたグラフ図である。FIG. 4 is a graph showing the EdU+/cTnT+ nuclei counted for the compounds picked up in the 1st screening of this example. 本実施例の１ｓｔ　ｓｃｒｅｅｎｉｎｇでピックアップした別の化合物に対するＥｄＵ＋／ｃＴｎＴ＋核数をカウントしたグラフ図である。FIG. 10 is a graph showing the number of EdU+/cTnT+ nuclei counted for another compound picked up in the 1st screening of this example. 本実施例の１ｓｔ　ｓｃｒｅｅｎｉｎｇでピックアップしたまた別の化合物に対するＥｄＵ＋／ｃＴｎＴ＋核数をカウントしたグラフ図である。FIG. 10 is a graph showing counts of EdU+/cTnT+ nuclei for yet another compound picked up in the 1st screening of this example. 本実施例のカウントの元となった免疫染色の一部を示す写真図である。FIG. 3 is a photographic diagram showing a part of immunostaining that was the basis for counting in this example. 本実施例の細胞数評価試験の方法を示す概略図である。It is a schematic diagram showing the method of the cell number evaluation test of the present example. 本実施例の細胞数評価試験の１日および１＋５日間培養後の明視野を示す写真図である。FIG. 2 is a photographic diagram showing a bright field after culturing for 1 day and 1+5 days in the cell number evaluation test of this example. 本実施例の各種ＡＬＫ５阻害剤を用いた試験のトータル細胞数の評価を示すグラフ図である。FIG. 4 is a graph showing evaluation of total cell count in tests using various ALK5 inhibitors of this example. 本実施例の各種ＡＬＫ５阻害剤を用いた試験のｃＴｎＴ（＋）％の評価を示すグラフ図である。FIG. 4 is a graph showing evaluation of cTnT(+)% in tests using various ALK5 inhibitors of this example. 本実施例の各種ＡＬＫ５阻害剤を用いた試験のｃＴｎＴ（＋）細胞数の評価を示すグラフ図である。FIG. 4 is a graph showing evaluation of cTnT(+) cell numbers in tests using various ALK5 inhibitors of this example. 心筋細胞の分化誘導の操作の一態様を示す模式図である。FIG. 2 is a schematic diagram showing one mode of operation for inducing differentiation of cardiomyocytes.
　以下、本発明に係る心筋細胞の増殖方法、心筋細胞の製造方法および心筋細胞増殖剤について、実施形態を示して説明する。ただし、本発明は以下の実施形態に限定されるものではない。 Hereinafter, the cardiomyocyte proliferation method, the cardiomyocyte production method, and the cardiomyocyte proliferation agent according to the present invention will be described with reference to embodiments. However, the present invention is not limited to the following embodiments.
　（心筋細胞の増殖方法）
　本実施形態の心筋細胞の増殖方法は、ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物の存在下で、心筋細胞を培養する工程を含む。 (Method for Proliferating Cardiomyocytes)
In the cardiomyocyte proliferation method of this embodiment, cardiomyocytes are cultured in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Including process.
　ＡＬＫ阻害剤（ＡＬＫインヒビター）は、ＡＬＫ（ａｃｔｉｖｉｎ　ｒｅｃｅｐｔｏｒ－ｌｉｋｅ　ｋｉｎａｓｅ）の機能を阻害する成分を広く指す。ＡＬＫ阻害剤は、例えば、核酸（ｓｉＲＮＡ、ｍｉＲＮＡ、およびアンチセンスオリゴヌクレオチドなど）、タンパク質、または低分子化合物である。 An ALK inhibitor (ALK inhibitor) broadly refers to a component that inhibits the function of ALK (activin receptor-like kinase). ALK inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds.
　前記ＡＬＫ阻害剤は、ＴＧＦβＲＩ（ＴＧＦ－β　ｒｅｃｅｐｔｏｒ　ｔｙｐｅ－１）（ＡＬＫ５）阻害剤であることが好ましい。また、前記ＡＬＫ５阻害剤は、ＬＹ３６４９４７、ＳＢ４３１５４２、Ａ８３－０１、およびＲｅｐＳｏｘからなる群より選択される１以上の化合物であることが好ましい。 The ALK inhibitor is preferably a TGFβRI (TGF-β receptor type-1) (ALK5) inhibitor. Also, the ALK5 inhibitor is preferably one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
　シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤は、シトクロムＰ４５０オメガ－ヒドロキシラーゼの機能を阻害する成分を広く指す。シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤は、例えば、核酸（ｓｉＲＮＡ、ｍｉＲＮＡ、およびアンチセンスオリゴヌクレオチドなど）、タンパク質、または低分子化合物である。シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤としては、１２（Ｓ）－ヒドロキシ－１６－ヘプタデシノイン酸であることが好ましい。 A cytochrome P450 omega-hydroxylase inhibitor broadly refers to a component that inhibits the function of cytochrome P450 omega-hydroxylase. Cytochrome P450 omega-hydroxylase inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds. A preferred cytochrome P450 omega-hydroxylase inhibitor is 12(S)-hydroxy-16-heptadecinoic acid.
　脂肪酸合成阻害剤は、脂肪酸の合成を阻害する成分を広く指す。脂肪酸合成阻害剤は、例えば、核酸（ｓｉＲＮＡ、ｍｉＲＮＡ、およびアンチセンスオリゴヌクレオチドなど）、タンパク質、または低分子化合物である。脂肪酸合成阻害剤としては、Ｃ７５、またはセルレニンであることが好ましい。 Fatty acid synthesis inhibitors broadly refer to ingredients that inhibit fatty acid synthesis. Fatty acid synthesis inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds. Preferably, the fatty acid synthesis inhibitor is C75 or cerulenin.
　ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、脂肪酸合成阻害剤からなる群より選択される１以上の化合物の使用量、すなわち、心筋細胞の増殖方法において用いる量は、後述する心筋細胞を培養する際に用いる場合、培地に対する濃度で０．５～４０μＭの範囲が好ましく、２～１０μＭがより好ましい。 ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. When it is used in the case where the concentration of the medium is 0.5 to 40 μM, preferably 2 to 10 μM.
　本実施形態において、心筋細胞とは、心筋の細胞を広く指し、具体的には、自己拍動の特性を有する細胞である。心筋細胞は、心筋前駆細胞も含む。また、心筋細胞は、拍動筋肉と電気伝導組織を形成する心筋細胞および血管平滑筋を生じる能力を有する細胞を広く含む。心筋細胞および心筋前駆細胞は互いに混在していても、それぞれ単独であってもよい。心筋細胞および心筋前駆細胞は、心筋マーカーである心筋トロポニン（ｃＴＮＴ）陽性、または、αミオシン重鎖（αＭＨＣ）陽性である細胞を広く含む。
　また、心筋細胞は、他の細胞種と比して心筋細胞の割合を多く含有した細胞集団も含む。好ましくは、心筋細胞を５０％以上有する細胞集団であり、好ましくは６０％または７０％以上、より好ましくは８０％以上、特に好ましくは９０％以上有する。 In the present embodiment, cardiomyocytes broadly refer to myocardial cells, specifically cells having self-pulsating properties. Cardiomyocytes also include myocardial progenitor cells. Cardiomyocytes also broadly include cells that have the potential to give rise to cardiomyocytes and vascular smooth muscle that form beating muscle and electrically conductive tissue. Cardiomyocytes and cardiomyocyte progenitor cells may be mixed with each other or may be individual. Cardiomyocytes and myocardial progenitor cells broadly include cells positive for the myocardial marker cardiac troponin (cTNT) or α-myosin heavy chain (αMHC).
Cardiomyocytes also include cell populations that contain a high percentage of cardiomyocytes relative to other cell types. A cell population containing 50% or more cardiomyocytes is preferred, preferably 60% or 70% or more, more preferably 80% or more, and particularly preferably 90% or more.
　前記心筋細胞は、多能性幹細胞由来の心筋細胞であることが好ましい。多能性幹細胞としては、ＥＳ細胞、ｎｔＥＳ細胞、ＧＳ細胞、ＥＧ細胞、Ｍｕｓｅ細胞、またはｉＰＳ細胞等が知られている。本実施形態の前記多能性幹細胞としては、ｉＰＳ細胞を用いることが好ましい。ｉＰＳ細胞は、体細胞から誘導される多能性幹細胞であり、分化多能性および自己複製能を有する。分化多能性とは、三胚葉系列すべてに分化できることを意味する。また、前記自己複製能とは、未分化状態を保持したまま増殖できる能力を意味する。 The cardiomyocytes are preferably cardiomyocytes derived from pluripotent stem cells. ES cells, ntES cells, GS cells, EG cells, Muse cells, iPS cells, and the like are known as pluripotent stem cells. iPS cells are preferably used as the pluripotent stem cells of the present embodiment. iPS cells are pluripotent stem cells derived from somatic cells and have pluripotency and self-renewal potential. Pluripotency means the ability to differentiate into all three germ layer lineages. Moreover, the self-renewal ability means the ability to proliferate while maintaining an undifferentiated state.
　また、前記多能性幹細胞は、ヒト由来の多能性幹細胞であることがより好ましい。さらに、ヒト由来のｉＰＳ細胞（Ｈｕｍａｎ　ｉＰＳ細胞、ＨｉＰＳ細胞、ヒトｉＰＳ細胞）であることが特に好ましい。 Also, the pluripotent stem cells are more preferably human-derived pluripotent stem cells. Furthermore, human-derived iPS cells (Human iPS cells, HiPS cells, human iPS cells) are particularly preferred.
　ｉＰＳ細胞を得る方法は、従来知られている方法により行うことができる。例えば、Ｋ．　Ｔａｋａｈａｓｈｉ　ａｎｄ　Ｓ．　Ｙａｍａｎａｋａ　（２００６）　Ｃｅｌｌ，　１２６：６６３－６７６、または、ＷＯ２００７／０６９６６６などに記載の手法を用いることができる。
　具体的には、特定の初期化因子をＤＮＡまたはタンパク質の形態で体細胞に導入することによって作成することができる。初期化因子としては、Ｏｃｔ３／４、Ｓｏｘ２、Ｓｏｘ１、Ｓｏｘ３、Ｓｏｘ１５、Ｓｏｘ１７、Ｋｌｆ４、Ｋｌｆ２、ｃ－Ｍｙｃ、Ｎ－Ｍｙｃ、Ｌ－Ｍｙｃ、Ｎａｎｏｇ、Ｌｉｎ２８、Ｆｂｘ１５、ＥＲａｓ、ＥＣＡＴ１５－２、Ｔｃｌ１、ｂｅｔａ－ｃａｔｅｎｉｎ、Ｌｉｎ２８ｂ、Ｓａｌｌ１、Ｓａｌｌ４、Ｅｓｒｒｂ、Ｎｒ５ａ２、Ｔｂｘ３またはＧｌｉｓ１等の遺伝子やコードするタンパク質を、単独または複数用いることができる。
　初期化因子がＤＮＡの場合は、ウイルス、プラスミド若しくは人工染色体などのベクター、リポフェクション、リポソーム、またはマイクロインジェクションなどを用いて細胞内に導入することができる。初期化因子がタンパク質の場合は、リポフェクション、細胞膜透過性ペプチドとの融合、またはマイクロインジェクションなどの手法によって細胞内に導入することができる。 A method for obtaining iPS cells can be performed by a conventionally known method. For example, K. Takahashi and S.; Yamanaka (2006) Cell, 126:663-676, or the method described in WO2007/069666 or the like can be used.
Specifically, it can be produced by introducing a specific reprogramming factor into a somatic cell in the form of DNA or protein. Reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERAs, ECAT15-2, Tcl1, Genes such as beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, or proteins encoding them can be used singly or in combination.
When the reprogramming factor is DNA, it can be introduced into cells using vectors such as viruses, plasmids or artificial chromosomes, lipofection, liposomes, or microinjection. When the reprogramming factor is a protein, it can be introduced into cells by techniques such as lipofection, fusion with a cell membrane permeable peptide, or microinjection.
　多能性幹細胞由来の心筋細胞を得る方法、すなわち、多能性幹細胞からの心筋細胞の分化誘導は、公知の方法により行うことができる。例えば、Ｔｏｈｙａｍａ，Ｓ．，ｅｔ　ａｌ．（２０１３）．Ｃｅｌｌ　Ｓｔｅｍ　Ｃｅｌｌ　１２（１）：１２７－１３７、Ｌａｆｌａｍｍｅ　ｅｔ　ａｌ．，　Ｎａｔ　Ｂｉｏｔｅｃｈ．　２００７　Ｓｅｐ；２５（９）：１０１５－２４、Ｕｏｓａｋｉ　ｅｔ　ａｌ．，　Ｐｌｏｓ　ＯＮＥ．　２０１１；６（８）：ｅ２３６５７に記載の手法を用いることができる。また、既成の心筋誘導培地や、それを含む既存のキット等を用いることもできる。 A method for obtaining pluripotent stem cell-derived cardiomyocytes, that is, induction of differentiation of cardiomyocytes from pluripotent stem cells can be performed by a known method. See, for example, Toyama, S.; , et al. (2013). Cell Stem Cell 12(1):127-137, Laflamme et al. , Nat Biotech. 2007 Sep;25(9):1015-24, Uosaki et al. , Plos ONE. 2011; 6(8): e23657 can be used. In addition, a ready-made myocardial induction medium, an existing kit containing the medium, or the like can also be used.
　具体的には、多能性幹細胞から心筋細胞へ分化誘導するために、種々の心筋分化誘導因子を用いることができる。心筋分化誘導因子としては、中胚葉誘導因子（アクチビンＡ、ＢＭＰ４、ｂＦＧＦ、ＶＥＧＦ、若しくはＳＣＦ等）、心臓決定因子（ＶＥＧＦ、またはＤＫＫ１等）、Ｗｎｔシグナル阻害剤（ＸＡＶ９３９、ＩＷＲ－１、ＩＷＰ－２、若しくはＩＷＰ－４等）、ＢＭＰシグナル阻害剤（ＮＯＧＧＩＮ等）、ＴＧＦβ／アクチビン／ＮＯＤＡＬシグナル阻害剤（ＳＢ４３１５４２等）、レチノイン酸シグナル阻害剤、または、心臓分化因子（例えば、ＶＥＧＦ、ｂＦＧＦ、若しくはＤＫＫ等）等が挙げられる。これらの心筋分化誘導因子は、単独または複数組み合わせて用いることができる。 Specifically, various myocardial differentiation-inducing factors can be used to induce the differentiation of pluripotent stem cells into cardiomyocytes. Cardiac differentiation inducers include mesoderm-inducing factors (activin A, BMP4, bFGF, VEGF, or SCF, etc.), cardiac determinants (VEGF, or DKK1, etc.), Wnt signal inhibitors (XAV939, IWR-1, IWP- 2, or IWP-4, etc.), BMP signal inhibitors (NOGGIN, etc.), TGFβ/activin/NODAL signal inhibitors (SB431542, etc.), retinoic acid signal inhibitors, or cardiac differentiation factors (e.g., VEGF, bFGF, or DKK, etc.). These myocardial differentiation-inducing factors can be used singly or in combination.
　多能性幹細胞から心筋細胞へ分化誘導する操作としては、まず、多能性幹細胞を培養し中胚葉へ分化を誘導する。中胚葉へ分化を誘導するには、前記中胚葉誘導因子の存在下で１～７日培養することで行うことができる。例えば、本実施形態ではＢＭＰ４の存在下で４～５日間培養することが好ましい。
　ついで、前記した心筋分化誘導因子の存在下で１～４日間培養を行う。例えば、本実施形態では心筋分化誘導因子の存在下で２～３日間培養することが好ましい。心筋分化誘導因子としては、内因性のＷｎｔ阻害物質であるＤｋｋ－１、またはＷｎｔシグナル阻害剤のＸＡＶ９３９、ＩＷＰ－４を用いることがさらに好ましい。これらの心筋分化誘導因子は併用してもよく、例えばＸＡＶ９３９、ＩＷＰ－４を併用してもよい。
　また、本実施形態では、中胚葉へ分化を誘導する前の前処理としてｍａｔｒｉｇｅｌの存在下で１～２日培養を行い、中胚葉へ分化を誘導する際にさらにｂＦＧＦの存在下で培養を行うことも好ましい。この操作により、より高効率で心筋細胞への分化誘導が行われ、中胚葉へ分化を誘導する培養の開始から４～１０日前後より、脈動する細胞が現れる。 As an operation for inducing differentiation from pluripotent stem cells to cardiomyocytes, first, pluripotent stem cells are cultured to induce differentiation into mesoderm. Differentiation into mesoderm can be induced by culturing for 1 to 7 days in the presence of the mesoderm-inducing factor. For example, in this embodiment, it is preferable to culture for 4 to 5 days in the presence of BMP4.
Then, culture is performed for 1 to 4 days in the presence of the cardiomyocyte differentiation-inducing factor. For example, in the present embodiment, culture is preferably performed for 2 to 3 days in the presence of cardiomyocyte differentiation-inducing factors. It is more preferable to use Dkk-1, which is an endogenous Wnt inhibitor, or XAV939 and IWP-4, which are Wnt signal inhibitors, as myocardial differentiation-inducing factors. These myocardial differentiation-inducing factors may be used in combination, for example, XAV939 and IWP-4 may be used in combination.
In the present embodiment, culture is performed in the presence of matrigel for 1 to 2 days as a pretreatment before inducing differentiation into mesoderm, and culture is further performed in the presence of bFGF when inducing differentiation into mesoderm. is also preferred. By this operation, cardiomyocyte differentiation is induced with higher efficiency, and pulsating cells appear from around 4 to 10 days after the initiation of culture for inducing differentiation into mesoderm.
　本実施形態の心筋細胞の増殖方法において、前記心筋細胞を、前記化合物のいずれか１以上の存在下で培養を行う工程を含む。例えば、化合物のいずれか１以上を添加した培地により培養を行うことが好ましい。培地には、上記成分に加えて他の成分を含んでいてもよい。 The cardiomyocyte proliferation method of the present embodiment includes the step of culturing the cardiomyocytes in the presence of any one or more of the compounds. For example, it is preferable to culture in a medium supplemented with any one or more of the compounds. The medium may contain other components in addition to the above components.
　心筋細胞を増殖するための培養は、前記分化誘導などにより得られた心筋細胞を分離し、改めてプレートに移植して培養を行ってもよい。または、前記分化誘導での培養を行ったプレートにおいて、改めて増殖するための培養を行ってもよい。
　本実施形態では、分化誘導で得られた心筋細胞を改めて低密度で播種し、心筋細胞を増殖するための培養を行うことが好ましい。播種の密度は、およそ０．１～５×１０４ｃｅｌｌｓ／ｃｍ２から適宜選択できる。好ましくは、０．５～２．０×１０４ｃｅｌｌｓ／ｃｍ２である。 Cultivation for proliferation of cardiomyocytes may be carried out by separating the cardiomyocytes obtained by the aforementioned induction of differentiation, transplanting them to a plate, and culturing them. Alternatively, culture for proliferation may be performed again on the plate that has been cultured for differentiation induction.
In this embodiment, the cardiomyocytes obtained by the induction of differentiation are preferably seeded again at a low density and cultured to proliferate the cardiomyocytes. The seeding density can be appropriately selected from approximately 0.1 to 5×10 4 cells/cm 2 . Preferably, it is 0.5 to 2.0×10 4 cells/cm 2 .
　心筋細胞を増殖するための培養は、従来知られた手段および条件で行うことができる。例えば、培養温度は３０～４０℃、好ましくは約３７℃、ＣＯ２濃度は約２％～５％、Ｏ２濃度はいわゆる低酸素条件、例えば約５％～２０％などが挙げられる。 Culture for proliferation of cardiomyocytes can be performed by conventionally known means and conditions. For example, the culture temperature is 30 to 40° C., preferably about 37° C., the CO 2 concentration is about 2% to 5%, and the O 2 concentration is so-called hypoxic conditions, eg about 5% to 20%.
　心筋細胞の培養に用いる培地は、特に限定されず、目的に応じて適宜選択することができる。例えば、基礎培地を用いることができる。基礎培地としては、例えばＩＭＤＭ培地、Ｍｅｄｉｕｍ　１９９培地、Ｅａｇｌｅ’ｓ　Ｍｉｎｉｍｕｍ　Ｅｓｓｅｎｔｉａｌ　Ｍｅｄｉｕｍ（ＥＭＥＭ）培地、α　ＭＥＭ培地、Ｄｕｌｂｅｃｃｏ’ｓ　ｍｏｄｉｆｉｅｄ　Ｅａｇｌｅ’ｓ　Ｍｅｄｉｕｍ（ＤＭＥＭ）培地、Ｈａｍ’ｓ　Ｆ１２培地、ＲＰＭＩ　１６４０培地、Ｆｉｓｃｈｅｒ’ｓ培地、Ｎｅｕｒｏｂａｓａｌ　Ｍｅｄｉｕｍ（ライフテクノロジーズ）、ＳｔｅｍＰｒｏ３４（Ｉｎｖｉｔｒｏｇｅｎ）、およびこれらを混合した混合培地などを用いてもよい。基礎培地は、血清を含有していても、無血清であってもよい。血清を含有する場合、５質量％～２０質量％の血清を含有する基礎培地を用いることができる。 The medium used for culturing cardiomyocytes is not particularly limited, and can be appropriately selected according to the purpose. For example, a basal medium can be used. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, α MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, and RPMI 1640 medium. , Fischer's medium, Neurobasal Medium (Life Technologies), StemPro34 (Invitrogen), and a mixed medium containing these may be used. The basal medium can be serum-containing or serum-free. When containing serum, a basal medium containing 5% to 20% by weight of serum can be used.
　培地は、他の成分を含んでいてもよい。他の成分としては、栄養素やそれ以外の添加物が挙げられる。栄養素としては、例えば血清代替物が挙げられる。血清代替物としては、アルブミン、トランスフェリン、Ｋｎｏｃｋｏｕｔ　Ｓｅｒｕｍ　Ｒｅｐｌａｃｅｍｅｎｔ（ＫＳＲ）（ＥＳ細胞培養時のＦＢＳの血清代替物）、Ｎ２サプリメント（Ｉｎｖｉｔｒｏｇｅｎ）、Ｂ２７サプリメント（Ｉｎｖｉｔｒｏｇｅｎ）、脂肪酸、インスリン、コラーゲン前駆体、微量元素、２－メルカプトエタノール、または１－チオールグリセロールなどを１種以上含んでいてもよい。他の成分としては、脂質、アミノ酸、Ｌ－グルタミン、Ｇｌｕｔａｍａｘ（Ｉｎｖｉｔｒｏｇｅｎ）、非必須アミノ酸、ビタミン、増殖因子、低分子化合物、抗生物質、抗酸化剤、ピルビン酸、緩衝剤、または無機塩類などを１種以上含んでいてもよい。 The medium may contain other ingredients. Other ingredients include nutrients and other additives. Nutrients include, for example, serum replacement. Serum substitutes include albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace amounts It may contain one or more elements such as 2-mercaptoethanol or 1-thiolglycerol. Other ingredients include lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, or inorganic salts. It may contain one or more kinds.
　本実施形態では、例えば培地としてＲＰＭＩ培地を用い、抗生物質としてインスリンの存在下で培養している。 In this embodiment, for example, RPMI medium is used as a medium and cultured in the presence of insulin as an antibiotic.
　本実施形態の心筋細胞の増殖方法で得られた心筋細胞は、ヒトの心臓の疾患（心疾患）の治療に用いることができる。ここで心臓の疾患とは、心臓における疾患、または心臓の処置の必要がある疾患を広く含む。本実施形態の心筋細胞を治療に用いるには、得られた心筋細胞を患者に投与する、または得られた心筋細胞を心臓の疾患の治療剤として用いてもよい。前記心筋細胞の投与の方法としては、細胞を液状に懸濁して心筋層に投与する、または細胞をシートやバンデージ等を介して添付するといった手段を用いることができる。前記心臓の疾患としては、心不全、虚血性心疾患、心筋梗塞、心筋症、心筋炎、肥大型心筋症、拡張相肥大型心筋症、拡張型心筋症などの疾患、または障害による欠損等を広く含み、心臓に影響のある疾患であれば限定されない。 The cardiomyocytes obtained by the cardiomyocyte proliferation method of the present embodiment can be used for treatment of human heart disease (cardiac disease). Here, the heart disease broadly includes diseases in the heart or diseases requiring treatment of the heart. In order to use the cardiomyocytes of this embodiment for treatment, the obtained cardiomyocytes may be administered to a patient, or the obtained cardiomyocytes may be used as a therapeutic agent for heart disease. As a method for administering the myocardial cells, a means of suspending the cells in a liquid and administering them to the myocardium, or attaching the cells via a sheet, bandage, or the like can be used. The heart disease includes heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic phase hypertrophic cardiomyopathy, dilated cardiomyopathy, etc., or defects due to disorders. It is not limited as long as it is a disease affecting the heart.
（心筋細胞の製造方法）
　本実施形態の心筋細胞の製造方法は、ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物の存在下で、心筋細胞を培養する工程を含む。前記化合物および心筋細胞を培養する工程については、前記心筋細胞の増殖方法において説明したものから選択できる。すなわち、本実施形態の心筋細胞の製造方法は、前記心筋細胞の増殖方法における工程を含んでいることが好ましい。
　換言すれば、培養中の心筋細胞に対して、前記心筋細胞の増殖方法における工程を加えることで、心筋細胞が増殖するので、増殖分の心筋細胞を製造することができる。 (Method for producing cardiomyocytes)
In the method for producing cardiomyocytes of this embodiment, cardiomyocytes are cultured in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Including process. The compound and the step of culturing cardiomyocytes can be selected from those described in the cardiomyocyte proliferation method. That is, the method for producing cardiomyocytes of the present embodiment preferably includes the steps in the method for growing cardiomyocytes.
In other words, by adding the steps in the method for growing cardiomyocytes to cultured cardiomyocytes, the cardiomyocytes proliferate, and the cardiomyocytes for the proliferation can be produced.
（心筋細胞増殖剤）
　本実施形態の心筋細胞増殖剤は、ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物を有効成分とする。前記化合物については、前記心筋細胞の増殖方法において説明したものから選択できる。 (cardiomyocyte proliferation agent)
The cardiomyocyte proliferation agent of this embodiment contains, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. The compound can be selected from those described in the cardiomyocyte proliferation method.
　本実施形態の心筋細胞増殖剤は、前記した心筋細胞の増殖方法および心筋細胞の製造方法に用いられるものであってもよい。すなわち、培養される心筋細胞を増殖するために添加されるものであってもよい。 The cardiomyocyte-proliferating agent of the present embodiment may be used in the above-described cardiomyocyte-proliferating method and cardiomyocyte-producing method. That is, it may be added to proliferate cultured cardiomyocytes.
　本実施形態の心筋細胞増殖剤を心筋細胞の増殖方法および心筋細胞の製造方法に用いる場合、培養中の心筋細胞に対して、その培養系に本実施形態の心筋細胞増殖剤を添加することで用いることができる。 When the cardiomyocyte-proliferating agent of the present embodiment is used in a cardiomyocyte-proliferating method and a cardiomyocyte-producing method, the cardiomyocyte-proliferating agent of the present embodiment is added to the culture system of cardiomyocytes in culture. can be used.
　また、別の態様として、心筋細胞増殖剤は、患者に投与されるものであってもよい。このとき、前記心筋細胞は、患者の体内のものであることが好ましい。特に、心臓の疾患を持つ、または心臓の処置の必要がある患者のものであることが好ましい。心臓の疾患または心臓の処置が必要となる状態については、前記心筋細胞の増殖方法で述べた例が挙げられる。 In another aspect, the cardiomyocyte proliferation agent may be administered to the patient. At this time, the cardiomyocytes are preferably those in the patient's body. In particular, it is preferred for patients with heart disease or in need of heart treatment. Cardiac diseases or conditions requiring treatment of the heart include the examples described in the cardiomyocyte proliferation method.
　このとき、心筋細胞増殖剤は、適宜他の成分を含んでいてもよい。例えば、心臓その他の循環器の疾患の処置に用いられる成分として従来知られているものを含んでいてもよい。
　また、ＡＬＫ阻害剤を含む心筋細胞増殖剤は、抗がんの治療効果のためにも用いられるものであってもよい。 At this time, the cardiomyocyte proliferation agent may contain other ingredients as appropriate. For example, it may contain ingredients conventionally known as those used in the treatment of diseases of the heart and other circulatory systems.
Cardiomyocyte proliferation agents, including ALK inhibitors, may also be used for anti-cancer therapeutic effects.
（本実施形態の効果）
　本実施形態の心筋細胞の増殖方法、心筋細胞の製造方法および心筋細胞増殖剤では、ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、または脂肪酸合成阻害剤を用いる。これらの化合物は、ヒト心筋細胞の増殖を誘導する効果を有する。 (Effect of this embodiment)
In the cardiomyocyte proliferation method, cardiomyocyte production method, and cardiomyocyte proliferation agent of the present embodiment, an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, or a fatty acid synthesis inhibitor is used. These compounds have the effect of inducing proliferation of human cardiomyocytes.
　本発明者らは、ヒト心筋細胞の増殖を誘導しうる化合物を見出すため、ヒトｉＰＳ細胞由来心筋細胞を用いて化合物スクリーニングを行った。その結果、ヒト心筋細胞を用いた化合物ライブラリースクリーニングでは、心筋増殖活性のある上記化合物群を見出した。同化合物は、約３ヶ月間の長期にわたる培養を行ったヒト心筋細胞においても２回以上の***を誘導することが可能であった。一方、同化合物に反応性を示さない心筋細胞もあり、現在遺伝子の網羅的発現解析により両者の差異の解明を進めている。また同化合物は、マウスＥＳ細胞由来心筋細胞に対しては、顕著な心筋増殖効果が認められなかったことから、ヒト心筋細胞特異的な増殖制御メカニズムの存在が示唆された。 The present inventors conducted compound screening using human iPS cell-derived cardiomyocytes in order to discover compounds that can induce proliferation of human cardiomyocytes. As a result, in compound library screening using human myocardial cells, the above compound group having myocardial proliferation activity was found. The same compound was able to induce mitosis twice or more even in human cardiomyocytes cultured for a long period of about 3 months. On the other hand, some cardiomyocytes do not show reactivity to the same compound, and we are currently clarifying the difference between the two by comprehensive gene expression analysis. In addition, the same compound did not exhibit a significant myocardial proliferative effect on mouse ES cell-derived cardiomyocytes, suggesting the existence of a human cardiomyocyte-specific growth control mechanism.
　心筋増殖に関する既報としては、ｐ３８阻害がｃｙｃｌｉｎＡ活性化を介して、ａｄｕｌｔ　ＣＭ　ｐｒｏｌｉｆｅｒａｔｉｏｎで行うこと、ｎｅｕｒｅｇｌｉｎ１がＥＲＢＢ２－ＥＲＢＢ４－ＥＲＫシグナルを介して行うこと、Ｈｉｐｐｏ　ｐａｔｈｗａｙがＹａｐ－ＰＩ３Ｋ－Ａｋｔ活性化、ついでＧＳＫ－３β不活性化、ＩＧＦ活性化により行うこと、ｍｉＲ－１５　ｆａｍｉｌｙ阻害がｐｒｏｌｏｎｇｅｄ　ｐｒｏｌｉｆｅｒａｔｉｏｎにより行うこと、Ｍｅｉｓ１がｄｅｌｅｔｉｏｎで、ＣＤＫ阻害剤（ｐ１５、ｐ１６、ｐ２１）の活性化を阻害して行うことなどが報告されている。
　しかし、従来、上記化合物群のＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、または脂肪酸合成阻害剤が、心筋細胞の増殖に関わっているという報告はない。 Previous reports on myocardial proliferation include p38 inhibition via cyclinA activation in adult CM proliferation, neureglin1 via ERBB2-ERBB4-ERK signaling, Hippo pathway via Yap-PI3K-Akt activation, and GSK-3β inactivation, by IGF activation, miR-15 family inhibition by prolonged proliferation, Meis1 by deletion, by inhibiting the activation of CDK inhibitors (p15, p16, p21) etc. have been reported.
However, there have been no reports that ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, or fatty acid synthesis inhibitors in the above compound groups are involved in cardiomyocyte proliferation.
　特に、ＡＬＫ阻害剤はヒト心筋細胞数を約１．５～２倍あまりに増加させることが見出された。ＡＬＫ阻害剤によく反応する心筋細胞では、３回の***が確認されている。すなわち、***前の８倍の細胞数への増殖に相当する。ＡＬＫ阻害剤の効果は、ヒト細胞特異的作用であり、マウス心筋細胞では効果が弱い。ＡＬＫ阻害剤の心筋細胞増殖制御に関しては、これまで全く報告がない。また、ＡＬＫ阻害剤には、すでに抗がん剤としての治験がなされている化合物があり、これを用いることで医薬品としての応用が可能である。 In particular, it was found that ALK inhibitors increased the number of human cardiomyocytes by about 1.5-2 fold. Three divisions have been confirmed in cardiomyocytes that respond well to ALK inhibitors. That is, it corresponds to proliferation to eight times the number of cells before division. The effects of ALK inhibitors are human cell-specific and weak in mouse myocardial cells. There has been no report on cardiomyocyte proliferation control by ALK inhibitors. In addition, among ALK inhibitors, there are compounds that have already been tested as anticancer agents, and their use as pharmaceuticals is possible.
　ヒトにおいて心筋細胞増殖を可能とし、心臓再生治療を実現することを目指す上ではヒト心筋細胞における明確な効果が必要であるが、本発明は上記化合物群のいずれか、すなわち単一の化合物においてそれを達成し、従来の問題を解決するポテンシャルを有している。
　本実施形態の心筋細胞の増殖方法、心筋細胞の製造方法および心筋細胞増殖剤は、心筋増殖および増殖刺激に対する反応性の分子機構を解明し、ヒト心筋細胞を増殖可能にすることにより、新しい心臓再生医療開拓への貢献が期待される。 In order to enable cardiomyocyte proliferation in humans and to realize cardiac regenerative therapy, a clear effect on human cardiomyocytes is necessary. and has the potential to solve conventional problems.
The method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the cardiomyocyte-proliferating agent of the present embodiment elucidate the molecular mechanism of myocardial proliferation and responsiveness to proliferation stimuli, and enable human cardiomyocytes to proliferate. It is expected to contribute to the development of regenerative medicine.
　以下、実施例によって本発明を具体的に説明するが、本発明は以下の記載によって限定されない。 The present invention will be specifically described below with reference to examples, but the present invention is not limited by the following description.
　＜心筋細胞の分化誘導＞
　ヒトｉＰＳ細胞より誘導した心筋細胞を用いて解析を行う必要性から、ヒトｉＰＳ－心筋細胞増殖スクリーニング系の確立を試みた。本発明者らは、２次元心筋細胞分化誘導法ＤＤ－ｐｒｏｔｏｃｏｌ（上述のＬａｆｌａｍｍｅ　ｅｔ　ａｌ，Ｎａｔ　Ｂｉｏｔｅｃｈ，　２００７など）に、ｄａｙ－１でのｍａｔｒｉｇｅｌ、ｄａｙ１－５でのｂＦＧＦ添加を加えることで、より効率良く心筋を分化する系（上述のＵｏｓａｋｉ　ｅｔ　ａｌ，Ｐｌｏｓ　ＯＮＥ，２０１１など）を確立している（図１９）。この方法では、ｄａｙ５－７でＲＰＭＩ培地＋Ｂ２７存在下でＤｋｋ－１を添加し、心筋細胞に分化している。
　この分化系は、さらに、あるマーカー（α型血小板由来増殖因子受容体；ＰＤＧＦＲα）を発現する細胞のみを純化するという操作を加えることもでき（Ｆｕｋｕｓｈｉｍａ　ｅｔ　ａｌ，ＰＬｏＳ　Ｏｎｅ．　２０２０　Ｎｏｖ　２；１５（１１）：ｅ０２４１２８７．）、以下の実施例ではこの純化操作を実施した。 <Differentiation induction of cardiomyocytes>
Because of the need to perform analysis using cardiomyocytes induced from human iPS cells, an attempt was made to establish a human iPS-cardiomyocyte proliferation screening system. The present inventors added matrigel on day-1 and bFGF on day 1-5 to the two-dimensional cardiomyocyte differentiation induction method DD-protocol (Laflamme et al, Nat Biotech, 2007, etc. described above), A system for more efficient myocardial differentiation (Uosaki et al, Plos ONE, 2011, etc. mentioned above) has been established (Fig. 19). In this method, Dkk-1 was added in the presence of RPMI medium+B27 on days 5-7 to differentiate into cardiomyocytes.
This differentiation system can be further manipulated to purify only cells expressing a certain marker (α-type platelet-derived growth factor receptor; PDGFRα) (Fukushima et al, PLoS One. 2020 Nov 2; 15 ( 11): e0241287.), this purification procedure was carried out in the examples below.
　本実施例で用いた心筋細胞の分化誘導の操作の概略を、時系列に則って図１に示す。ｉＰＳ細胞は、京都大学　山中伸弥教授より提供を受けたヒトｉＰＳ細胞（２０１Ｂ６）を用いた。ｉＰＳ細胞に対して、ｄａｙ　－２またはｄａｙ　－３からｃｏｎｄｉｔｉｏｎｅｄ　ｍｅｄｉｕｍ培養中でｂＦＧＦ（添加量：４ｎｇ／ｍＬ，Ｆｉｎａｌ　Ｃｏｎｃ．）を添加、ｄａｙ　－１からｄａｙ０までｍａｔｒｉｇｅｌ（添加量：培地の１／６０）をさらに添加した。ｄａｙ０－５まではＲＰＭＩ培地＋Ｂ２７　ｉｎｓｕｌｉｎ（－）中で培養を行い、ｄａｙ０－１まではＡｃｔｉｖｉｎＡ、ｄａｙ１－５まではＢＭＰ４（添加量：１ｎｇ／ｍＬ，Ｆｉｎａｌ　Ｃｏｎｃ．）とｂＦＧＦ（添加量：１０ｎｇ／ｍＬ，Ｆｉｎａｌ　Ｃｏｎｃ．）を添加した。ｄａｙ５にＰＤＧＦＲαを発現する細胞のみを純化、ＲＰＭＩ培地＋Ｂ２７（添加量：ＲＰＭＩに対して２％）にｉｎｓｕｌｉｎ（＋）中で再培養をおこない、ｄａｙ５－７ではＷｎｔシグナル阻害剤（ＸＡＶ９３９、ＩＷＰ４）をさらに添加した。 Fig. 1 shows an outline of the operation for inducing the differentiation of cardiomyocytes used in this example in chronological order. Human iPS cells (201B6) provided by Professor Shinya Yamanaka of Kyoto University were used as iPS cells. bFGF (addition amount: 4 ng/mL, Final Conc.) was added to the iPS cells in conditioned medium culture from day -2 or day -3, and matrigel (addition amount: 1/1 of the medium) was added from day -1 to day 0. 60) was further added. Culture was performed in RPMI medium + B27 insulin (-) until day 0-5, Activin A until day 0-1, and BMP4 (addition amount: 1 ng/mL, Final Conc.) and bFGF (addition amount: 10 ng) until day 1-5. /mL, Final Conc.) was added. Only cells expressing PDGFRα were purified on day 5, recultured in insulin (+) in RPMI medium + B27 (addition amount: 2% relative to RPMI), and Wnt signal inhibitors (XAV939, IWP4) on day 5-7. was further added.
　この系では、培養開始時のｉＰＳ細胞が、ｄａｙ５からｍｅｓｏｄｅｒｍ（中胚葉）細胞となり、ｄａｙ９前後からは脈動する細胞が現れ、培養により心筋細胞が得られた。
　図２に、この系により得られた細胞の心筋トロポニンＴの発現率（ｃＴｎＴ（＋）％）を示す。この分化系により、ｃＴｎＴ（＋）％が９７％を超える心筋細胞が得られることが示された（図２左側のグラフ）。この心筋細胞をより低密度で再びプレートに播種し、これを用いて以後の解析を行った（図２右側の模式図）。 In this system, iPS cells at the start of culture became mesoderm cells from day 5, pulsating cells appeared from around day 9, and cardiomyocytes were obtained by culture.
FIG. 2 shows the cardiac troponin T expression rate (cTnT(+)%) of cells obtained by this system. It was shown that this differentiation system yields cardiomyocytes with a cTnT(+)% of over 97% (graph on the left side of FIG. 2). The cardiomyocytes were plated again at a lower density and used for subsequent analysis (schematic diagram on the right side of FIG. 2).
　＜解析条件の検討＞
　まず、分化のどの地点の心筋を用いるのか、心筋の播種条件、解析条件等の設定を行い、解析の条件を検討した。
　ポジティブコントロールの選定のための増殖評価（特許文献１より）の模式図を図３に示す。増殖評価のポジティブコントロールとして、本発明者らがマウス心筋細胞の増殖スクリーニング系で見出した化合物を参考に設定した。ＥＳ細胞より誘導したマウス心筋細胞、および、ｅｍｂｒｙｏ、ｎｅｏｎａｔｅならびにａｄｕｌｔの各マウスから単離したマウス心筋細胞を用いて、各種キナーゼ阻害剤（図中のＺＭ３３６３７２、ＳＵ１４９８、ＫＮ６２、ＫＮ９３、ＢＩＯ、ＣＨＩＲ、ＳＢ２０３５８０）による心筋増殖解析を行った。なお、ＣＨＩＲはＣＨＩＲ９９０２１を意味する。 <Examination of analysis conditions>
First, we set the myocardial seeding conditions, analysis conditions, etc., and examined the analysis conditions.
FIG. 3 shows a schematic diagram of proliferation evaluation (from Patent Document 1) for selection of a positive control. As a positive control for proliferation evaluation, a compound found by the present inventors in a mouse cardiomyocyte proliferation screening system was set as a reference. Mouse myocardial cells derived from ES cells and mouse myocardial cells isolated from embryo, neonate and adult mice were treated with various kinase inhibitors (ZM336372, SU1498, KN62, KN93, BIO, CHIR, SB203580) performed myocardial proliferation analysis. In addition, CHIR means CHIR99021.
　Ｍｏｕｓｅ　ＥＳ－ｃａｒｄｉｏｍｙｏｃｙｔｅ（ｍＥＳＣＭ）に前記各化合物を加え培養した。図４にＤＭＳＯ（コントロール）の他ＢＩＯ、ＳＵ１４９８、ＫＮ９３投与培養の細胞染色を示した。ＢＩＯ、ＳＵ１４９８、ＫＮ９３には写真中で暗色の心筋細胞（ｃＴｎＴ）に、写真中で明色であるＥｄＵ、ＤＡＰＩの染色が見られた。図５にＳ期（ＥｄＵ－ｐｏｓｉｔｉｖｅ　ＣＭ）、図６にＭ期（ｐＨ３すなわちｐｈｏｓｐｈｏ－ｈｉｓｔｏｎｅ　ｐｏｓｉｔｉｖｅ）の細胞量を示した。図５、図６はそれぞれＤＭＳＯ群に対する＊Ｐ＜０．０５を示した（ｎ＝３のＤｕｎｎ　ｔｅｓｔ）。これらの結果から、ＢＩＯ、ＳＵ１４９８、ＫＮ９３には心筋増殖能が見られた。
　同様に検討した結果をまとめると、ＧＳＫ３β阻害剤であるＢＩＯ、ＣＨＩＲ９９０２１、ｐ３８ＭＡＰＫ阻害剤（ｐ３８ｉ）であるＳＢ２０３５８０、およびＥＲＫ　ａｃｔｉｖａｔｏｒであるＳＵ１４９８に心筋増殖能が見られた。 Each compound was added to Mouse ES-cardiomyocyte (mESCM) and cultured. FIG. 4 shows cell staining of DMSO (control), BIO, SU1498, and KN93-administered cultures. In BIO, SU1498, and KN93, cardiomyocytes (cTnT) dark in the photograph were stained with EdU and DAPI, which were bright in the photograph. FIG. 5 shows the cell amount in S phase (EdU-positive CM), and FIG. 6 shows the cell amount in M phase (pH 3, ie, phospho-histone positive). Figures 5 and 6 each showed *P<0.05 against the DMSO group (Dunn test of n=3). From these results, BIO, SU1498 and KN93 were found to have myocardial proliferation ability.
Summarizing the results of similar studies, BIO and CHIR99021, which are GSK3β inhibitors, SB203580, which is a p38MAPK inhibitor (p38i), and SU1498, which is an ERK activator, were found to have myocardial proliferation ability.
　続いて、これらの化合物をヒトｉＰＳ－心筋細胞に添加したところ、それぞれ単剤では、心筋増殖効果がわずかに見られるのみであった（図示せず）。一方、これらの化合物を併用して添加することで、後述するように、スクリーニングでの評価基準ＥｄＵ＋／ｃＴｎＴ＋細胞核数が比較的安定して１．５倍を超える結果が得られた。
　ただし、ＢＩＯはヒトｉＰＳ－心筋細胞に対し、細胞毒性を示すため、その上流（図３）にあるＷｎｔ３ａを用いることとした。
　したがって、以後のスクリーニング系では、ポジティブコントロールとして、Ｗｎｔ３ａ、ＣＨＩＲ９９０２１、ｐ３８ｉ（ＳＢ２０３５８０）、およびＳＵ１４９８の４剤を併用して用いた。 Subsequent addition of these compounds to human iPS-cardiomyocytes resulted in only modest myocardial proliferative effects of each single agent (not shown). On the other hand, by adding these compounds in combination, the number of EdU+/cTnT+ cell nuclei, which is the criteria for evaluation in screening, was relatively stable and exceeded 1.5 times, as described later.
However, since BIO exhibits cytotoxicity to human iPS-cardiomyocytes, it was decided to use Wnt3a upstream of BIO (FIG. 3).
Therefore, in subsequent screening systems, Wnt3a, CHIR99021, p38i (SB203580), and SU1498 were used in combination as positive controls.
　＜ヒト心筋細胞の増殖＞
　分化のどの地点の心筋を用いるのか、心筋の播種条件、解析条件等の設定を行った。
　心筋細胞の増殖の流れを図７に示した。ｈｕｍａｎ　ｉＰＳ細胞より誘導した心筋細胞（ｄ１９）を低密度条件（１．３３ｘ１０４ｃｅｌｌｓ／ｃｍ２）で播種した。血清存在下に培養２４ｈｒｓ後、心筋細胞がプレートに接着しているのを確認し、前記ポジティブコントロールの各化合物を目的の濃度で添加した。２日間培養後、ＥｄＵを２０μＭ添加した。さらに１日間培養後、細胞の固定を行い、心筋細胞のマーカーであるｃａｒｄｉａｃ　ｔｒｏｐｏｎｉｎＴ（ｃＴｎＴ）で免疫染色を行った後、ｉｎ　Ｃｅｌｌ　Ａｎａｌｙｚｅｒ　６０００を用いて細胞の撮影を行った。 <Proliferation of human cardiomyocytes>
Settings such as which point of differentiation to use myocardium, myocardium seeding conditions, analysis conditions, etc. were performed.
FIG. 7 shows the flow of proliferation of cardiomyocytes. Cardiomyocytes (d19) induced from human iPS cells were seeded under low-density conditions (1.33×10 4 cells/cm 2 ). After culturing in the presence of serum for 24 hours, adhesion of cardiomyocytes to the plate was confirmed, and each compound of the positive control was added at a desired concentration. After culturing for 2 days, 20 μM of EdU was added. After culturing for another day, the cells were fixed and immunostained with cardiac troponin T (cTnT), which is a cardiomyocyte marker.
　代表的な写真を図８に示した。ｃＴｎＴ（写真中では暗色）が発現した心筋細胞内に、ＥｄＵ、ＤＡＰＩ（写真中では明色）で示されていたが、ネガティブコントロールにはｃＴＮＴおよびわずかなＥｄＵのシグナル、ポジティブコントロールには、ｃＴＮＴ、より多いＥｄＵおよびＤＡＰＩのシグナル（それぞれ矢印）が検出された。
　この画像について、解析ソフト（Ｄｅｖｅｌｏｐｅｒ　Ｔｏｏｌｂｏｘ　１．９）を用いてＥｄＵ＋／ｃＴｎＴ＋の細胞核（画像上白色矢印）の数をカウントし、評価を行った結果を図９に示した。ポジティブコントロールの添加にて、ＥｄＵ＋／ｃＴｎＴ＋細胞核数は１．５倍をこえて増大したことが示された。 A representative photograph is shown in FIG. EdU and DAPI (light color in the photograph) were shown in cardiomyocytes in which cTnT (dark color in the photograph) was expressed. , more EdU and DAPI signals (arrows, respectively) were detected.
For this image, the number of EdU+/cTnT+ cell nuclei (white arrows in the image) was counted using analysis software (Developer Toolbox 1.9), and the evaluation results are shown in FIG. Addition of the positive control showed a more than 1.5-fold increase in the number of EdU+/cTnT+ cell nuclei.
　＜ヒト心筋細胞の増殖＞
　条件が整った後、Ｃａｙｍａｎ　Ｂｉｏ－ａｃｔｉｖｅ　ｌｉｐｉｄ　ｓｃｒｅｅｎｉｎｇ　ｌｉｂｒａｒｙ　Ｉ＆ＩＩの解析を行った。
　Ｂｉｏ－ａｃｔｉｖｅ　ｌｉｐｉｄ　ｓｃｒｅｅｎｉｎｇ　ｌｉｂｒａｒｙ　Ｉは、スクリーニング対象の７８５以上の化合物が９６ウェルのマトリックスチューブに１．０ｍＭ　ＤＭＳＯ内に保存されているものを用いた。Ｂｉｏ－ａｃｔｉｖｅ　ｌｉｐｉｄ　ｓｃｒｅｅｎｉｎｇ　ｌｉｂｒａｒｙ　ＩＩは、スクリーニング対象の１８５以上の化合物が９６ウェルのマトリックスチューブに０．１ｍＭ　ＤＭＳＯ内に保存されているものを用いた。 <Proliferation of human cardiomyocytes>
After the conditions were established, Cayman Bio-active lipid screening library I & II were analyzed.
Bio-active lipid screening library I used more than 785 compounds to be screened stored in 96-well matrix tubes in 1.0 mM DMSO. Bio-active lipid screening library II used 96-well matrix tubes containing >185 compounds to be screened in 0.1 mM DMSO.
　その他のスクリーニングの条件は以下のように行った。
　細胞播種条件：
　・ｈｉＰＳ細胞より分化誘導した心筋を用いた。
　・細胞播種密度　１．３３ｘ１０４ｃｅｌｌｓ／ｃｍ２
　・細胞播種の翌日（２０ｈｒｓ後）にｒｅａｇｅｎｔを添加した。
　評価条件：
　・ｃＴｎＴ（＋）／ＥｄＵ（＋）　ｎｕｃｌｅａｒ　ｎｕｍｂｅｒｓ
　・化合物添加、３ｄａｙｓ（７２ｈｒｓ）後に固定し、染色した。
　・ＥｄＵ　取り込み　２４ｈｒｓで評価した。
　化合物の条件：
　・Ｃａｙｍａｎ　Ｂｉｏ　ａｃｔｉｖｅ　ｌｉｐｉｄ　Ｉ＆ＩＩ　ｓｃｒｅｅｎｉｎｇ　ｌｉｂｒａｒｙ
　　ｐｌａｔｅ　１－１１；　１０μＭ，　２μＭ（ｓｔｏｃｋ；１．０ｍＭ）
　　ｐｌａｔｅ　１２－１４；　１μＭ，　０．２μＭ（ｓｔｏｃｋ；０．１ｍＭ） Other screening conditions were as follows.
Cell seeding conditions:
・Myocardial muscle induced to differentiate from hiPS cells was used.
・Cell seeding density 1.33×10 4 cells/cm 2
- Reagent was added on the day after cell seeding (20 hrs later).
Evaluation conditions:
・cTnT(+)/EdU(+) nuclear numbers
-Fixed and stained 3 days (72 hrs) after the addition of the compound.
-EdU uptake was evaluated at 24 hrs.
Compound conditions:
・Cayman Bioactive lipid I & II screening library
plate 1-11; 10 μM, 2 μM (stock; 1.0 mM)
plate 12-14; 1 μM, 0.2 μM (stock; 0.1 mM)
　１ｓｔ　ｓｃｒｅｅｎｉｎｇ：
　・各条件１ｗｅｌｌずつ設定した。
　・１ｗｅｌｌにつき６視野撮影した。
　・６視野カウントを行い、ｎ＝６とし値をグラフ化した。
　ついで、
　・ＥｄＵ＋／ｃＴｎＴ＋　ｎｕｃｌｅａｒ　ｎｕｍｂｅｒｓの値で評価した。
　・値がｎｅｇａｔｉｖｅ　ｃｏｎｔｒｏｌの１．５倍を超えたものをｐｉｃｋ　ｕｐした。
　２ｎｄ　ｓｃｒｅｅｎｉｎｇ
　・各条件３ｗｅｌｌずつ設定した。
　・１ｗｅｌｌにつき６視野撮影した。
　・１８（３ｘ６）視野カウントを行い、ｎ＝１８とし値をグラフ化した。
　ついで、
　・ＥｄＵ＋／ｃＴｎＴ＋　ｎｕｃｌｅａｒ　ｎｕｍｂｅｒｓの値で評価した。
　・ｎｅｇａｔｉｖｅ　ｃｏｎｔｒｏｌに比べ、有意に増大したものをｐｉｃｋ　ｕｐした。 1st screening:
・One well was set for each condition.
・Six fields of view were photographed per well.
- 6 fields of view were counted, and the values were graphed with n=6.
Then,
- Evaluated by EdU+/cTnT+ nuclear numbers.
- Those whose values exceeded 1.5 times the negative control were picked up.
2nd screen
- Three wells were set for each condition.
・Six fields of view were photographed per well.
- 18 (3 x 6) visual field counts were performed and the values were graphed with n = 18.
Then,
- Evaluated by EdU+/cTnT+ nuclear numbers.
- Those that increased significantly compared to the negative control were picked up.
　スクリーニングの結果、１ｓｔ　ｓｃｒｅｅｎｉｎｇでは、ｈｉｔした化合物数は、Ｃａｙｍａｎ化合物９８２化合物中、１０３化合物であった。
　次に、２ｎｄ　ｓｃｒｅｅｎｉｎｇでは、解析を行った２濃度でともに有意に増大を示した化合物は、３化合物であった。この３化合物は、
　Ｒ２２９０－０９８：１２（Ｓ）－ｈｙｄｒｏｘｙ－１６－Ｈｅｐｔａｄｅｃｙｎｏｉｃ　Ａｃｉｄ
　Ｒ２２９０－３５８：Ｃ７５
　Ｒ２２９０－３７０：Ｃｅｒｕｌｅｎｉｎ
　であった。 As a result of screening, the number of hit compounds in the 1st screening was 103 out of 982 Cayman compounds.
Next, in the 2nd screening, 3 compounds showed a significant increase at the 2 concentrations analyzed. These three compounds are
R2290-098: 12(S)-hydroxy-16-Heptadecynoic Acid
R2290-358: C75
R2290-370: Cerulenin
Met.
　１ｓｔ　ｓｃｒｅｅｎｉｎｇでピックアップした化合物に対し、Ｉ；１０μＭ，２μＭ　ＩＩ；１μＭ，０．２μＭの２濃度で添加し、同様に解析を行った。ＥｄＵ＋／ｃＴｎＴ＋　ｎｕｃｌｅａｒ　ｎｕｍｂｅｒｓをカウントし、グラフ化した。結果を図１０、図１１、図１２に示す。
　図１０に示すように、Ｒ２２９０－０９８：１２（Ｓ）－ｈｙｄｒｏｘｙ－１６－Ｈｅｐｔａｄｅｃｙｎｏｉｃ　Ａｃｉｄ、および、Ｒ２２９０－３５８：Ｃ７５については、２濃度を添加した場合のいずれも有意に増大した。Ｒ２２９０－３７０：Ｃｅｒｕｌｅｎｉｎについては、１濃度（２μＭ）でのみ有意に増大が見られた。
　また、これら３化合物とターゲットを同じくする化合物で、一方の添加濃度で増殖中の心筋細胞核数が有意に増大したものが見いだされた（図１１、図１２）。それぞれ、
　Ｒ２２８９－９４９：ＬＹ３６４９４７
　Ｒ２２８９－８８０：ＳＢ４３１５４２
　であった。
　これらも含めた心筋増殖効果が認められた５化合物について、さらに解析を行った。 Two concentrations of I; 10 μM, 2 μM II; 1 μM, 0.2 μM were added to the compounds picked up in the 1st screening, and analyzed in the same manner. EdU+/cTnT+ nuclear numbers were counted and graphed. The results are shown in FIGS. 10, 11 and 12. FIG.
As shown in FIG. 10, both R2290-098:12(S)-hydroxy-16-Heptadecynoic Acid and R2290-358:C75 significantly increased when two concentrations were added. R2290-370: For Cerulenin, a significant increase was seen only at one concentration (2 μM).
In addition, it was found that one of the compounds with the same target as these three compounds significantly increased the number of proliferating cardiomyocyte nuclei (FIGS. 11 and 12). Respectively,
R2289-949: LY364947
R2289-880: SB431542
Met.
Further analysis was performed on the 5 compounds that were recognized to have myocardial proliferation effects, including these.
　図１３に、上述のＥｄＵ＋／ｃＴｎＴ＋細胞核数のカウントの元となった免疫染色写真の代表的なものを示す。写真からも、各化合物（Ｃ７５，ＬＹ３６４９４７，１２（Ｓ）－ｈｙｄｒｏｘｙ－１６－Ｈｅｐａｔａｄｅｃｙｎｏｉｃ　ａｃｉｄ，Ｃｅｒｕｌｅｎｉｎ，ＳＢ４３１５４２）の添加により、ＥｄＵ＋／ｃＴｎＴ＋　ｎｕｃｌｅａｒ　ｎｕｍｂｅｒｓ（明色のシグナル）がｎｅｇａｔｉｖｅ　ｃｏｎｔｒｏｌに比べて増大傾向にあることが示された。 FIG. 13 shows typical immunostained photographs from which the EdU+/cTnT+ cell nuclei were counted above. From the photograph, addition of each compound (C75, LY364947, 12(S)-hydroxy-16-Hepatadecynoic acid, Cerulenin, SB431542) tends to increase the EdU+/cTnT+ nuclear numbers (bright signal) compared to the negative control. was shown to be in
　＜心筋細胞数による評価＞
　上記心筋細胞増殖ｓｃｒｅｅｎｉｎｇでは、評価の指標としてＥｄＵ＋／ｃＴｎＴ＋　ｎｕｃｌｅａｒ　ｎｕｍｂｅｒｓを用いていた。ＥｄＵ＋／ｃＴｎＴ＋は、ＥｄＵを添加していた間（２４ｈｒｓ）にＤＮＡ合成をおこなっていた心筋細胞を示す。ヒトの心筋細胞は、マウス等のげっ歯類の心筋細胞と異なり、大部分が１核の細胞であるが、***中で一時的に２核となっている細胞（ｂｉ－ｎｕｃｌｅａｒ　ｃｅｌｌ）等もあり、ＥｄＵ＋／ｃＴｎＴ＋ｎｕｃｌｅａｒ　ｎｕｍｂｅｒｓの増大は必ずしも心筋細胞数の増大と一致しているとは言えない。そこで、心筋細胞数で評価を行う系の条件設定を行った。 <Evaluation by cardiomyocyte count>
In the above cardiomyocyte proliferation screening, EdU+/cTnT+ nuclear numbers were used as evaluation indices. EdU+/cTnT+ indicates cardiomyocytes undergoing DNA synthesis during the period of EdU addition (24 hrs). Unlike the cardiomyocytes of rodents such as mice, human cardiomyocytes are mostly mononuclear cells, but there are also binuclear cells that are temporarily dividing and have binuclear cells. Therefore, an increase in EdU+/cTnT+nuclear numbers does not necessarily correspond to an increase in the number of myocardial cells. Therefore, we set up conditions for a system that evaluates the number of myocardial cells.
　評価方法の概略を図１４に示す。ｓｃｒｅｅｎｉｎｇ時と同様、低密度でｈｉＰＳより分化誘導した心筋細胞を播種（０．９３×１０４ｃｅｌｌｓ／ｃｍ２）し、細胞接着を確認後、上記の各化合物を添加した。１日後、および１＋５日後に、心筋細胞数を数えた。あわせて、ｃＴＮＴ　ＦＡＣＳ解析を行った。 An outline of the evaluation method is shown in FIG. Cardiomyocytes induced to differentiate from hiPS were seeded at a low density (0.93×10 4 cells/cm 2 ) in the same manner as during screening, and after confirming cell adhesion, each of the above compounds was added. Cardiomyocyte numbers were counted after 1 day and 1+5 days. In addition, cTNT FACS analysis was performed.
　図１５に、１日および１＋５日間培養後の明視野写真を示した。図中のネガティブコントロールではｃＴｎＴ（＋）値は９３．８％、ポジティブコントロールではｃＴｎＴ（＋）値は９７．５％であった。ポジティブコントロールの添加で、心筋細胞が増大傾向にあることが見受けられる。 Fig. 15 shows bright field photographs after 1 day and 1+5 days of culture. The negative control in the figure had a cTnT(+) value of 93.8%, and the positive control had a cTnT(+) value of 97.5%. It can be seen that cardiomyocytes tend to increase with the addition of the positive control.
　ついで、各種のＡＬＫ５阻害剤を添加し、心筋細胞数が増大するかを解析した。
　ＬＹ３６４９４７、ＳＢ４３１５４２はともにＡＬＫ５阻害剤であり、上述したように、心筋細胞数の増大がみられる。この心筋細胞数の増大は、ＡＬＫ５シグナルを阻害することによるものである可能性がある。そのため、他のＡＬＫ５阻害剤であるＡ８３－０１、ＲｅｐＳｏｘを用いて解析を行った。Ａ８３－０１は、ＡＬＫ５の強力な阻害剤で、ＡＬＫ４、７も阻害することで知られる。一方、ＲｅｐＳｏｘ（２－（３－（６－ｍｅｔｈｙｌｐｙｒｉｄｉｎ－２－ｙｌ）－１Ｈ－ｐｙｒａｚｏｌ－４－ｙｌ）－１，５－ｎａｐｈｔｈｙｒｉｄｉｎｅ）（図１６～１８において「ＴＧＦｂＲ　ＫＩ」と示される）は、ＡＬＫ５選択的な阻害剤である。 Next, various ALK5 inhibitors were added to analyze whether the cardiomyocyte number increased.
Both LY364947 and SB431542 are ALK5 inhibitors and, as mentioned above, increase the number of myocardial cells. This increase in cardiomyocyte number may be due to inhibition of ALK5 signaling. Therefore, analysis was performed using other ALK5 inhibitors, A83-01 and RepSox. A83-01 is a potent inhibitor of ALK5 and is also known to inhibit ALK4,7. On the other hand, RepSox (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) (denoted as “TGFbR KI” in FIGS. 16-18) ALK5 selective inhibitor.
　各ＡＬＫ５阻害剤について、添加後のトータル細胞数を図１６、ｃＴｎＴ（＋）％を図１７、ｃＴｎＴ（＋）の細胞数を図１８に示した。ｎ＝３～９である。このうち、特にＡ８３－０１の添加で、心筋細胞数が有意に増大した。ＳＢ４３１５４２と同等にＲｅｐＳｏｘ（ＴＧＦｂＲ　ＫＩと表記）も心筋細胞数を増加させた。
　これらの結果から、ＡＬＫ５シグナルを阻害することが、心筋細胞数の増大につながること、ＡＬＫ５阻害剤が心筋細胞数を増やす化合物として、薬剤に使用できる可能性が示唆された。 For each ALK5 inhibitor, the total cell number after addition is shown in FIG. 16, cTnT(+)% is shown in FIG. 17, and the cTnT(+) cell number is shown in FIG. n=3-9. Among these, addition of A83-01 in particular significantly increased the number of myocardial cells. Similar to SB431542, RepSox (denoted as TGFbR KI) also increased cardiomyocyte number.
These results suggested that inhibition of ALK5 signaling leads to an increase in the number of myocardial cells, and the possibility that ALK5 inhibitors can be used as drugs as compounds that increase the number of myocardial cells.
　本発明の心筋細胞の増殖方法、心筋細胞の製造方法および心筋細胞増殖剤は、薬剤によりヒト心筋細胞の増殖の誘導を可能とし、心臓再生医療に寄与することができる。 The method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the agent for proliferating cardiomyocytes of the present invention enable the induction of human cardiomyocyte proliferation with drugs, and can contribute to cardiac regenerative medicine.

Claims (18)

  1. 　ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物の存在下で、心筋細胞を培養する工程を含む、心筋細胞の増殖方法。 A method for growing cardiomyocytes, comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  2. 　前記化合物がＡＬＫ阻害剤である、請求項１に記載の心筋細胞の増殖方法。 The method for proliferating cardiomyocytes according to claim 1, wherein the compound is an ALK inhibitor.
  3. 　前記ＡＬＫ阻害剤がＴＧＦβＲＩ（ＡＬＫ５）阻害剤である、請求項２に記載の心筋細胞の増殖方法。 The cardiomyocyte proliferation method according to claim 2, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.
  4. 　前記ＡＬＫ５阻害剤が、ＬＹ３６４９４７、ＳＢ４３１５４２、Ａ８３－０１、およびＲｅｐＳｏｘからなる群より選択される１以上の化合物である、請求項３に記載の心筋細胞の増殖方法。 The cardiomyocyte proliferation method according to claim 3, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  5. 　前記心筋細胞が多能性幹細胞由来の心筋細胞である、請求項１～４のいずれか一項に記載の心筋細胞の増殖方法。 The method for growing cardiomyocytes according to any one of claims 1 to 4, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.
  6. 　前記多能性幹細胞がヒト由来である、請求項１～５のいずれか一項に記載の心筋細胞の増殖方法。 The method for growing cardiomyocytes according to any one of claims 1 to 5, wherein the pluripotent stem cells are derived from humans.
  7. 　ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物の存在下で、心筋細胞を培養する工程を含む、心筋細胞の製造方法。 A method for producing cardiomyocytes, comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  8. 　前記化合物がＡＬＫ阻害剤である、請求項７に記載の心筋細胞の製造方法。 The method for producing cardiomyocytes according to claim 7, wherein the compound is an ALK inhibitor.
  9. 　前記ＡＬＫ阻害剤がＴＧＦβＲＩ（ＡＬＫ５）阻害剤である、請求項８に記載の心筋細胞の製造方法。 The method for producing cardiomyocytes according to claim 8, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.
  10. 　前記ＡＬＫ５阻害剤が、ＬＹ３６４９４７、ＳＢ４３１５４２、Ａ８３－０１、およびＲｅｐＳｏｘからなる群より選択される１以上の化合物である、請求項９に記載の心筋細胞の製造方法。 The method for producing cardiomyocytes according to claim 9, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  11. 　前記心筋細胞が多能性幹細胞由来の心筋細胞である、請求項７～１０のいずれか一項に記載の心筋細胞の製造方法。 The method for producing cardiomyocytes according to any one of claims 7 to 10, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.
  12. 　前記多能性幹細胞がヒト由来である、請求項７～１１のいずれか一項に記載の心筋細胞の製造方法。 The method for producing cardiomyocytes according to any one of claims 7 to 11, wherein the pluripotent stem cells are derived from humans.
  13. 　ＡＬＫ阻害剤、シトクロムＰ４５０オメガ－ヒドロキシラーゼ阻害剤、および脂肪酸合成阻害剤からなる群より選択される１以上の化合物を有効成分とする、心筋細胞増殖剤。 A cardiomyocyte proliferation agent containing, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  14. 　前記化合物がＡＬＫ阻害剤である、請求項１３に記載の心筋細胞増殖剤。 The cardiomyocyte proliferation agent according to claim 13, wherein the compound is an ALK inhibitor.
  15. 　前記ＡＬＫ阻害剤がＴＧＦβＲＩ（ＡＬＫ５）阻害剤である、請求項１４に記載の心筋細胞増殖剤。 The cardiomyocyte proliferation agent according to claim 14, wherein the ALK inhibitor is a TGFβRI (ALK5) inhibitor.
  16. 　前記ＡＬＫ５阻害剤が、ＬＹ３６４９４７、ＳＢ４３１５４２、Ａ８３－０１、およびＲｅｐＳｏｘからなる群より選択される１以上の化合物である、請求項１５に記載の心筋細胞増殖剤。 The cardiomyocyte proliferation agent according to claim 15, wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  17. 　前記心筋細胞が多能性幹細胞由来の心筋細胞である、請求項１３～１６のいずれか一項に記載の心筋細胞増殖剤。 The cardiomyocyte proliferation agent according to any one of claims 13 to 16, wherein the cardiomyocytes are cardiomyocytes derived from pluripotent stem cells.
  18. 　前記多能性幹細胞がヒト由来である、請求項１３～１７のいずれか一項に記載の心筋細胞増殖剤。 The cardiomyocyte proliferation agent according to any one of claims 13 to 17, wherein the pluripotent stem cells are derived from humans.
PCT/JP2022/009718 2021-03-08 2022-03-07 Method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent WO2022191130A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2023505544A JPWO2022191130A1 (en) 2021-03-08 2022-03-07

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202163157834P 2021-03-08 2021-03-08
US63/157,834 2021-03-08

Publications (1)

Publication Number Publication Date
WO2022191130A1 true WO2022191130A1 (en) 2022-09-15

Family

ID=83226653

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2022/009718 WO2022191130A1 (en) 2021-03-08 2022-03-07 Method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent

Country Status (2)

Country Link
JP (1) JPWO2022191130A1 (en)
WO (1) WO2022191130A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130331389A1 (en) * 2012-06-11 2013-12-12 National Cheng Kung University Methods and Compositions for Cardiomyocyte Replenishment by Endogenous and Progenitor Stem Cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130331389A1 (en) * 2012-06-11 2013-12-12 National Cheng Kung University Methods and Compositions for Cardiomyocyte Replenishment by Endogenous and Progenitor Stem Cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
COHEN ETHAN DAVID, YEE MIN, PORTER GEORGE A., RITZER ERIN, MCDAVID ANDREW N., BROOKES PAUL S., PRYHUBER GLORIA S., O’REILLY MICHAE: "Neonatal hyperoxia inhibits proliferation and survival of atrial cardiomyocytes by suppressing fatty acid synthesis", JCI INSIGHT, vol. 6, no. 5, 8 March 2021 (2021-03-08), XP055967804, DOI: 10.1172/jci.insight.140785 *
JIANG SHAN, HUO DAN, WANG XUEYING, ZHAO HUAN, TAN JIANG, ZENG QINGHUA, O'ROURKE STEPHEN, SUN CHENGWEN: "beta-adrenergic Receptor-stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 w-hydroxylase", JOURNAL OF CARDIOVASCULAR PHARMACOLOGY, RAVEN PRESS, NEW YORK, NY, US, vol. 70, no. 2, 1 August 2017 (2017-08-01), US , pages 94 - 101, XP009539641, ISSN: 0160-2446, DOI: 10.1097/FJC.0000000000000499 *
MATTHEW P. PAPPAS; LINDSAY N. PEIFER; SUNNY S. K. CHAN: "Dual TGFβ and Wnt inhibition promotes Mesp1‐mediated mouse pluripotent stem cell differentiation into functional cardiomyocytes", DEVELOPMENT GROWTH AND DIFFERENTIATION., RICHMOND, VIC. : BLACKWELL PUBL. ASIA, US, vol. 62, 4 November 2020 (2020-11-04), US , pages 487 - 494, XP071144350, ISSN: 0012-1592, DOI: 10.1111/dgd.12694 *
MOHAMED TAMER M.A., ANG YEN-SIN, RADZINSKY ETHAN, ZHOU PING, HUANG YU, ELFENBEIN ARYE, FOLEY AMY, MAGNITSKY SERGEY, SRIVASTAVA DEE: "Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration", CELL, ELSEVIER, AMSTERDAM NL, vol. 173, no. 1, 1 March 2018 (2018-03-01), Amsterdam NL , pages 104 - 116.e12, XP055967738, ISSN: 0092-8674, DOI: 10.1016/j.cell.2018.02.014 *

Also Published As

Publication number Publication date
JPWO2022191130A1 (en) 2022-09-15

Similar Documents

Publication Publication Date Title
JP7023820B2 (en) Cortical interneurons and other neuronal cells generated by directing the differentiation of pluripotent cells and pluripotent cells
AU2014279065B2 (en) Method for producing renal precursor cells, and drug containing renal precursor cells
JP7458012B2 (en) Method for inducing differentiation from intermediate mesoderm cells to renal progenitor cells, and method for inducing differentiation from pluripotent stem cells to renal progenitor cells
Xu et al. A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species
US10857185B2 (en) Compositions and methods for reprogramming non-neuronal cells into neuron-like cells
Kim et al. Efficient specification of interneurons from human pluripotent stem cells by dorsoventral and rostrocaudal modulation
CN104694570B (en) For generating the combinatorial chemistry genetic method of the multipotential stem cell of induction
KR102029391B1 (en) Method of producing multipotent stem cells
US20150250824A1 (en) Methods and compositions for expansion of stem cells and other cells
JP6316938B2 (en) Method for preparing induced neural stem cells reprogrammed from non-neuronal cells using HMGA2
TWI438275B (en) Method for promoting differentiation of stem cell into insulin producing cell
JP6351018B2 (en) Stem cell differentiation induction method using amino acid composition modified medium, and medium kit containing the medium
US10612002B2 (en) Human pluripotent stem cell derived endocardial endothelium
EP2907870A1 (en) Reprogramming peptide and use thereof
KR102133621B1 (en) Transplantation adjuvant in cell therapy using neural progenitor cells
KR101539132B1 (en) Method for induction cardiac myocyte from somatic cells using direct reprogramming strategy by small molecule treatment without genetic modification
WO2022191130A1 (en) Method for proliferating cardiomyocytes, method for producing cardiomyocytes, and cardiomyocyte proliferating agent
JP7437766B2 (en) Method for producing pluripotent stem cells that are free from mesendoderm differentiation resistance
Shan et al. Polycomb group protein Bmi1 is required for the neuronal differentiation of mouse induced pluripotent stem cells
Jin Engineering Strategies for Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes
KR101768581B1 (en) Generation of Reprogramming Cell Line from Single Cells of spontaneously Immortalized Somatic cell
Baio Microgravity Exerts an Age-Dependent Effect on Cardiovascular Progenitor Cell Development
Raciti Reprogramming fibroblasts to Neural-Stem-like cells by structured overexpression of pallial patterning genes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22767086

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2023505544

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22767086

Country of ref document: EP

Kind code of ref document: A1